TWI868333B - A method and drug for treating Alzheimer's disease - Google Patents

Abstract

The present invention relates to a method and a drug for preventing and treating Alzheimer's disease, comprising administering a therapeutically effective amount of a fibrinolytic enzyme activation pathway component to a subject. The present invention also relates to a drug, a drug composition, a product, and a reagent kit containing the fibrinolytic enzyme activation pathway component for treating the above-mentioned disease.

Description

A method and drug for treating Alzheimer's disease

The present invention relates to a method for preventing or treating Alzheimer's disease, comprising administering an effective amount of a component of a fibrinolytic enzyme activation pathway or a compound related thereto, such as fibrinolytic enzyme, to a subject to improve clinical symptoms and signs.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by global dementia such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes. The cause is still unknown. The main manifestations are cognitive decline, mental symptoms and behavioral disorders, and a gradual decline in daily living ability. It is divided into three stages according to the degree of deterioration of cognitive ability and physical function. The first stage, usually 1 to 3 years, is a mild dementia stage. The symptoms are memory loss, with a prominent forgetfulness of recent events; decreased judgment ability, the patient cannot analyze, think, or judge events, and has difficulty dealing with complex problems; carelessness at work or housework, inability to independently carry out shopping, financial affairs, etc., and social difficulties; although still able to do some familiar daily tasks, they are confused and puzzled by new things, emotionally indifferent, occasionally irritable, and often suspicious; time orientation disorder occurs, and the patient can orient himself to the place and people he is in, but has difficulty orienting himself to the geographical location, and has poor visual-spatial ability for complex structures; he has a small vocabulary and difficulty naming. The second stage, usually 2 to 10 years, is the moderate dementia stage. The symptoms are severe damage to both long- and short-term memory, decreased visual-spatial ability of simple structures, and disorientation to time and place; severe impairment in problem solving and identifying similarities and differences between things; inability to independently carry out outdoor activities, requiring help with dressing, personal hygiene, and maintaining personal appearance; inability to calculate; various neurological symptoms, including aphasia, apraxia, and agnosia; emotional changes from indifference to irritability, frequent walking, and urinary incontinence. The third stage, usually 8 to 12 years, is the severe dementia stage. The patient is completely dependent on the caregiver, has severe memory loss, and only retains fragmentary memories; cannot take care of himself in daily life, has incontinence, presents with mutism, limb rigidity, and a positive pyramidal fasciculus sign is found on physical examination, with primitive reflexes such as strong grasping, groping, and sucking. Eventually, he falls into a coma and usually dies from complications such as infection.

The current treatment method is mainly symptomatic treatment to control the psychopathological symptoms associated with Alzheimer's disease. For example, anti-anxiety drugs are used for anxiety, agitation, and insomnia; anti-depressants are used for depression; antipsychotic drugs are used to control the patient's behavioral disorders. In addition, in order to improve cognitive function and delay the progression of the disease, nootropics or drugs that improve cognitive function are used, such as drugs that act on neurotransmitters, cerebral vasodilators, and brain metabolism-boosting drugs. For the treatment of Alzheimer's disease, other treatment methods and drugs need to be found.

The present invention has found that fibrosinogen can promote memory recovery in patients with Alzheimer's disease, improve cognitive ability, significantly reduce and alleviate the clinical symptoms and signs of Alzheimer's disease, and prevent and treat Alzheimer's disease.

Specifically, the present invention relates to the following items:

1. In one aspect, the present application relates to a method for preventing and treating Alzheimer's disease, comprising administering to a subject with Alzheimer's disease a therapeutically effective amount of one or more compounds selected from the following: components of a fibrinolytic enzyme activation pathway, compounds that can directly activate fibrinolytic enzymes or indirectly activate fibrinolytic enzymes by activating upstream components of the fibrinolytic enzyme activation pathway, compounds that mimic the activity of fibrinolytic enzymes or fibrinolytic enzymes, compounds that can upregulate the expression of fibrinolytic enzymes or fibrinolytic enzyme activators, fibrinolytic enzyme analogs, fibrinolytic enzyme analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.

On the one hand, the present application relates to the use of one or more compounds selected from the following in the preparation of a medicament for treating Alzheimer's disease, wherein the one or more compounds are selected from: a component of a fibrinolytic enzyme activation pathway, a compound capable of directly activating fibrinolytic enzyme or indirectly activating fibrinolytic enzyme by activating an upstream component of the fibrinolytic enzyme activation pathway, a compound that mimics the activity of fibrinolytic enzyme or fibrinolytic enzyme, a compound capable of upregulating the expression of fibrinolytic enzyme or fibrinolytic enzyme activator, a fibrinolytic enzyme analog, a fibrinolytic enzyme analog, a tPA or uPA analog, and an antagonist of a fibrinolytic inhibitor.

On the one hand, the present application relates to a drug or drug composition for treating Alzheimer's disease comprising one or more compounds selected from the following: components of the fibrinolytic enzyme activation pathway, compounds that can directly activate fibrinolytic enzymes or indirectly activate fibrinolytic enzymes by activating upstream components of the fibrinolytic enzyme activation pathway, compounds that mimic the activity of fibrinolytic enzymes or fibrinolytic enzymes, compounds that can upregulate the expression of fibrinolytic enzymes or fibrinolytic enzyme activators, fibrinolytic enzyme analogs, fibrinolytic enzyme analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.

2. The method, use, drug or drug composition of item 1, wherein the component of the fibrinolytic enzyme activation pathway is selected from fibrinolytic enzyme, recombinant human fibrinolytic enzyme, Lys-fibrinolytic enzyme, Glu-fibrinolytic enzyme, fibrinolytic enzyme, fibrinolytic enzyme and fibrinolytic enzyme variants and analogs containing one or more kringle domains and protease domains of fibrinolytic enzyme and fibrinolytic enzyme , mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA and uPA.

3. The method, use, drug or drug composition of item 1, wherein the antagonist of the fibrinolytic inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, such as an antibody.

4. The method, use, drug or drug composition of any one of items 1-3, wherein the compound has one or more effects on Alzheimer's disease subjects selected from the following: promoting the degradation of amyloid protein Aβ40 or Aβ42 in brain tissue, improving memory function, improving cognitive ability, improving geographical location recognition ability, relieving anxiety or depressive symptoms, reducing Aβ42 deposition in brain tissue, promoting the degradation of Tau protein in brain tissue, promoting the cleavage of Pro-BDNF in brain tissue to form mature BDNF, promoting the expression of BDNF in brain tissue, promoting the cleavage of Pro-NGF in brain tissue to form mature NGF, and improving hippocampus damage in brain tissue.

5. The method, use, drug or drug composition of any one of items 1-4, wherein the compound is fibronectin.

6. The method, use, drug or drug composition of any one of items 1-5, wherein the fibronectin is human full-length fibronectin or a conservatively substituted variant thereof.

7. The method, use, drug or drug composition of any one of items 1-5, wherein the fibronectin has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has the lysine binding activity or proteolytic activity of fibronectin.

8. The method, use, drug or drug composition of any one of items 1-5, wherein the profibrinolytic enzyme comprises a protein having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with sequence 14 and still having the proteolytic activity of profibrinolytic enzyme.

9. The method, use, drug or drug composition of any one of items 1 to 5, wherein the fibrinolysinogen is selected from Glu-fibrinolysinogen, Lys-fibrinolysinogen, mini-fibrinolysinogen, micro-fibrinolysinogen, delta-fibrinolysinogen or variants thereof that retain the proteolytic activity of fibrinolysinogen.

10. The method, use, drug or drug composition of any one of items 1-5, wherein the fibrolytic enzyme comprises the amino acid sequence shown in sequence 2, 6, 8, 10, 12 or a conservatively substituted variant of the amino acid sequence shown in sequence 2, 6, 8, 10, 12.

11. The method, use, drug or drug composition of any one of items 1-10, wherein the compound is used in combination with one or more other therapeutic methods or drugs.

12. The method, use, drug or drug composition of claim 11, wherein the other treatment methods include cell therapy (including stem cell therapy), supportive therapy and physical therapy.

13. The method, use, drug or drug composition of claim 11, wherein the other drug is other drug for treating Alzheimer's disease.

14. The method, use, drug or drug composition of any one of items 1-13, wherein the compound is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery) or intramuscular administration.

In any of the above embodiments of the present invention, the profibrinolytic enzyme may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12, and still have profibrinolytic enzyme activity, such as lysine binding activity or proteolytic activity. In some embodiments, the profibrinolytic enzyme is a protein having 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, and still having profibrinolytic enzyme activity, such as lysine binding activity or proteolytic activity.

In some embodiments, the fibrinolysinogen is a protein comprising a fibrinolysinogen active fragment and still having fibrinolysinogen activity, such as proteolytic activity. In some embodiments, the fibrinolysinogen is selected from Glu-fibrinolysinogen, Lys-fibrinolysinogen, minifibrinolysinogen, microfibrinolysinogen, delta-fibrinolysinogen or a variant thereof retaining fibrinolysinogen activity, such as proteolytic activity. In some embodiments, the fibrinolysinogen is a natural or synthetic human fibrinolysinogen, or a variant or fragment thereof still retaining fibrinolysinogen activity, such as lysine binding activity or proteolytic activity. In some embodiments, the fibrinolysinogen is a human fibrinolysinogen ortholog from primates or rodents or a variant or fragment thereof that still retains fibrinolysinogen activity, such as lysine binding activity or proteolytic activity. In some embodiments, the amino acids of the fibrinolysinogen are shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the fibrinolysinogen is human native fibrinolysinogen.

In some embodiments, the subject is a human. In some embodiments, the subject lacks or is deficient in profibrinolytic enzyme. In some embodiments, the deficiency or deficiency is congenital, secondary and/or local.

In some embodiments, the drug composition comprises a pharmaceutically acceptable carrier and a fibronectin for use in the aforementioned method. In some embodiments, the kit may be a preventive or therapeutic kit comprising: (i) fibronectin for use in the aforementioned method and (ii) means for delivering the fibronectin to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or instructions for use, which indicates that the fibronectin is administered to the subject to implement any of the aforementioned methods.

In some embodiments, the article of manufacture comprises: a container comprising a label; and comprising (i) fibrosinogen or a pharmaceutical composition comprising fibrosinogen for use in the aforementioned method, wherein the label indicates that the fibrosinogen or composition is administered to the subject to perform any of the aforementioned methods.

In some embodiments, the kit or product further comprises one or more additional components or containers containing other drugs.

In some embodiments of the aforementioned method, the fibrinolysinogen is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, subcutaneous administration of fibrinolysinogen for treatment. In some embodiments of the aforementioned method, the fibrinolysinogen is administered in combination with an appropriate polypeptide carrier or stabilizer. In some embodiments of the aforementioned method, the fibrolytic enzyme is administered at a dose of 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg, 10-100 mg/kg (calculated per kilogram of body weight) or 0.0001-2000 mg/cm2, 0.001-800 mg/cm2, 0.01-600 mg/cm2, 0.1-400 mg/cm2, 1-200 mg/cm2, 1-100 mg/cm2, 10-100 mg/cm2 (calculated per square centimeter of body surface area) per day, preferably repeated at least once, preferably at least daily.

The present invention explicitly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been explicitly disclosed in this application, just as the above-mentioned technical solutions have been individually and explicitly disclosed. In addition, the present invention also explicitly covers the combination between various embodiments and their elements, and the technical solutions after these combinations are explicitly disclosed in this article.

Fibrinolytic system, also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the fibrinolysis process, mainly including fibrinolytic enzymes (fibrinolysinogen), fibrinolysin, fibrinolysin activators, and fibrinolysis inhibitors. Fibrinolysin activators include tissue-type fibrinolysin activator (t-PA) and urokinase-type fibrinolysin activator (u-PA). t-PA is a serine protease synthesized by vascular endothelial cells. t-PA activates fibrinogen, and this process mainly takes place on fibrin; urokinase-type fibrinogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells, and can directly activate fibrinogen without the need for fibrin as a cofactor. Fibrinogen (PLG) is synthesized by the liver. When blood coagulates, PLG is adsorbed on the fibrin network in large quantities. Under the action of t-PA or u-PA, it is activated into fibrinolytic enzyme, which promotes the dissolution of fibrin. Fibrinolytic enzyme (PL) is a serine protease, which has the following functions: degrades fibrin and fibrinogen; hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc.; converts fibrinogen into fibrinolytic enzyme; hydrolyzes tocopherols, etc. Fibrinolytic inhibitors: including fibrinolytic activator inhibitor (PAI) and α2 antifibrinolytic enzyme (α2-AP). PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG. α2-AP is synthesized by the liver and binds to PL in a 1:1 ratio to form a complex, inhibiting PL activity; FⅩⅢ allows α2-AP to bind to fibrin with a covalent bond, reducing the sensitivity of fibrin to the action of PL. Substances that inhibit the activity of the fibrinolytic system in the body: PAI-1, complement C1 inhibitor; α2 antifibrinolytic enzyme; α2 macroglobulin.

The term "component of the fibronectin activation pathway" of the present invention covers:

1. Fibrinolysinogen, Lys-fibrinolysinogen, Glu-fibrinolysinogen, micro-plasminogen, delta-fibrinolysinogen; their variants or analogs;

2. Fibrolytic enzymes and their variants or analogs; and

3. Fibrinogen activators, such as tPA and uPA, and tPA or uPA variants and analogs comprising one or more domains of tPA or uPA (such as one or more kringle domains and a proteolytic domain).

"Variants" of the above-mentioned fibrinolysinogen, fibrinolysin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as proteins that still have fibrinolysinogen, fibrinolysin, tPA or uPA activity by adding, deleting and/or substituting, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids. For example, "variants" of fibrinolysin, fibrinolysin, tPA, and uPA include mutant variants of these proteins obtained by, e.g., 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acid substitutions.

The "fibrinolytic enzyme variants" of the present invention encompass proteins that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12, and still have fibrinolytic enzyme activity, such as lysine binding activity or proteolytic activity. For example, the "fibrinolytic enzyme variant" of the present invention can be a protein with 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, and still having fibrinolytic enzyme activity, such as lysine binding activity or proteolytic activity. Specifically, the lysinogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as mutant variants of these proteins obtained by conservative amino acid substitutions, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid.

The fibrinolysin of the present invention may be a human fibrinolysin ortholog from a primate or rodent, or a variant thereof that still retains fibrinolysin activity, such as lysine binding activity or proteolytic activity, such as the fibrinolysin shown in SEQ ID NO: 2, 6, 8, 10 or 12, such as the human native fibrinolysin shown in SEQ ID NO: 2.

The "analogs" of the above-mentioned fibrinolysinogen, fibrinolysin, tPA and uPA include compounds that provide substantially similar effects to fibrinolysinogen, fibrinolysin, tPA or uPA, respectively.

The above-mentioned "variants" and "analogs" of plasminogen, plasmin, tPA and uPA cover plasminogen, plasmin, tPA and uPA "variants" and "analogs" comprising one or more domains (e.g., one or more kringle domains and a proteolytic domain). For example, "variants" and "analogs" of plasminogen cover plasminogen variants and analogs comprising one or more plasminogen domains (e.g., one or more kringle domains and a proteolytic domain), such as mini-plasminogen. "Variants" and "analogs" of plasmin encompass plasmin "variants" and "analogs" that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain), such as mini-plasmin and delta-plasmin.

Whether the above-mentioned "variants" or "analogs" of fibrinolysinogen, fibrinolysin, tPA or uPA have the activity of fibrinolysinogen, fibrinolysin, tPA or uPA, respectively, or whether they provide substantially similar effects to fibrinolysinogen, fibrinolysin, tPA or uPA, respectively, can be detected by methods known in the art, for example, by measuring the level of activated fibrinolysin activity based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence activated cell sorting method), for example, it can be measured by referring to the method selected from the following references: Ny, A., Leonardsson, G., Hagglund, A.C., Hagglof, P., Ploplis, V.A., Carmeliet, P. and Ny, T. (1999). Ovulation in plasminogen-deficient mice. Endocrinology 140, 5030-5035; Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). "Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator". J. Clin. Invest. 74 (5): 1625–33; Gravanis I, Tsirka SE (February 2008). "Tissue-type plasminogen activator as a therapeutic target in stroke". Expert Opinion on Therapeutic Targets. 12 (2): 159–70; Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). "Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo". Blood. 74 (2): 722–8.

In some embodiments of the present invention, the "component of the fibronectin activation pathway" of the present invention is fibronectin. In some embodiments, the fibronectin is a human full-length fibronectin or a conservative substitution variant thereof that retains fibronectin activity (e.g., its lysine binding activity and proteolytic activity). In some embodiments, the fibronectin is selected from Glu-fibronectin, Lys-fibronectin, mini-fibronectin, micro-fibronectin, delta-fibronectin or variants thereof that retain fibronectin activity (e.g., its lysine binding activity or proteolytic activity). In some embodiments, the fibrinolysinogen is a natural or synthetic human fibrinolysinogen, or a conservative substitution variant or fragment thereof that still retains fibrinolysinogen activity (e.g., its lysine binding activity or proteolytic activity). In some embodiments, the fibrinolysinogen is a human fibrinolysinogen ortholog from a primate or rodent, or a conservative substitution variant or fragment thereof that still retains fibrinolysinogen activity. In some embodiments, the fibrinolysinogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the fibrinolysinogen comprises a conservative substitution sequence of an amino acid sequence as shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the amino acids of the fibrinolysinogen are as shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the fibrinolysinogen is a conservative substitution variant of the fibrinolysinogen shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the fibrinolysinogen is a human natural fibrinolysinogen or a conservative mutant thereof. In some embodiments, the fibrinolysinogen is a human natural fibrinolysinogen or a conservative substitution variant thereof as shown in sequence 2.

"Compounds capable of directly activating fibrinogen or indirectly activating fibrinogen by activating upstream components of the fibrinogen activation pathway" refers to any compound capable of directly activating fibrinogen or indirectly activating fibrinogen by activating upstream components of the fibrinogen activation pathway, such as tPA, uPA, streptokinase, saseloprepase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplasase, pamiplase, staphylokinase.

The "antagonist of fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks or prevents the action of fibrinolysis inhibitor, such as PAI-1, cytochrome C1 inhibitor, α2 antifibrinolytic enzyme and α2 macroglobulin. The antagonists are, for example, antibodies to PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, or antisense RNA or small RNA that blocks or downregulates the expression of, for example, PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin but have no PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin function, or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin.

Fibrinolytic enzymes are key components of the fibrinolytic enzyme activation system (PA system). They are broad-spectrum proteases that can hydrolyze several components of the extracellular matrix (ECM), including fibroin, gelatin, fibronectin, laminin, and proteoglycans. In addition, fibrinolytic enzymes can activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, fibrinolytic enzymes are considered to be an important upstream regulator of extracellular proteolysis. Fibrinolytic enzymes are formed by proteolysis of fibrinolytic enzymes by two physiological PAs: tissue-type fibrinolytic enzyme activator (tPA) or urokinase-type fibrinolytic enzyme activator (uPA). Due to the relatively high levels of fibrinolysins in plasma and other body fluids, it has been traditionally believed that the PA system is regulated primarily through the synthesis and activity levels of PAs. The synthesis of the PA system components is tightly regulated by different factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of fibrinolysins and PAs. The major inhibitor of fibrinolysins is α2-antiplasmin. The activity of PAs is also regulated by the fibrinolysinogen activator inhibitor-1 (PAI-1) for both uPA and tPA, and the lysozyme activator inhibitor-2 (PAI-2) which primarily inhibits uPA. Certain cells have a specific cell surface receptor for uPA (uPAR) that has direct hydrolytic activity.

Plasminogen is a single-chain glycoprotein composed of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is mainly synthesized in the liver and is present in large quantities in the extracellular fluid. The plasma content of plasminogen is approximately 2 μM. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamic acid and is therefore called Glu-plasminogen. However, in the presence of plasminogen, Glu-plasminogen is hydrolyzed at Lys76-Lys77 to Lys-plasminogen. Compared with glutamate-fibrinolysinogen, lysine-fibrinolysinogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of both forms of fibrinolysinogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked two-chain protease fibrinolysin. The amino-terminal part of fibrinolysinogen contains five homologous tricyclic rings, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain lysine binding sites that mediate the specific interaction of fibrinolysinogen with fibrin and its inhibitor α2-AP. A newly discovered 38 kDa fragment of fibrinolysinogen, which includes kringles1-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, can be generated by proteolysis of fibrinolysinogen by several proteases.

The main substrate of fibrinolytic enzymes is fibrin, and the dissolution of fibrin is the key to preventing pathological thrombosis. Fibrinolytic enzymes also have substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that fibrinolytic enzymes also play an important role in ECM reconstruction. Indirectly, fibrinolytic enzymes can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3, and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that fibrinolytic enzymes may be an important upstream regulator of extracellular proteolysis. In addition, fibrinolytic enzymes have the ability to activate certain latent forms of growth factors. In vitro, fibrinolytic enzymes can also hydrolyze components of the complement system and release proteolytic fragments.

"Fibrinolytic enzyme" is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.

"Fillinogen" is the zymogen form of fibrinolysin. According to the sequence in Swiss prot, it is composed of 810 amino acids and has a molecular weight of about 90kD, calculated according to the amino acid sequence of natural human fibrinolysin containing a signal peptide (SEQ ID 4). It is a glycoprotein mainly synthesized in the liver and can circulate in the blood. The cDNA sequence encoding the amino acid sequence is shown in SEQ ID 3. The full-length fibrinolysin contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5). With reference to the sequence in swiss prot, its signal peptide includes residues Met1-Gly19, PAp includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, Kringle4 includes residues Cys377-Cys454, and Kringle5 includes residues Cys481-Cys560. According to NCBI data, the serine protease domain includes residues Val581-Arg804.

Glu-plasminogen is the natural full-length plasminogen of human beings, consisting of 791 amino acids (without the 19-amino acid signal peptide). The cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. In vivo, there is also a Lys-plasminogen formed by hydrolysis of the 76th-77th amino acids of Glu-plasminogen, as shown in SEQ ID NO: 6, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 5. Delta-plasminogen (δ-plasminogen) is a fragment of the full-length plasminogen that lacks the Kringle2-Kringle5 structure and contains only Kringle1 and the serine protease domain (also known as the protease domain (PD)). The amino acid sequence of delta-plasminogen has been reported in the literature (SEQ ID NO: 8), and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 7. Mini-plasminogen is composed of Kringle5 and serine protease domains. Literature reports that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid). Its amino acid sequence is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9. Micro-plasminogen only contains a serine protease domain. Some literature reports that its amino acid sequence includes residues Ala543-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid), and some patent document CN102154253A reports that its sequence includes residues Lys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid). In this patent application, the sequence of micro-plasminogen refers to patent document CN102154253A, and its amino acid sequence is shown in SEQ ID NO: 12. The cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 11.

The structure of full-length plasminogen is also described in the article by Aisina et al. (Aisina RB, Mukhametova LI. Structure and function of plasminogen/plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605). In this article, Aisina et al. described that fibrinolysinogen includes Kringle1, 2, 3, 4, 5 domains and serine protease domain (also known as protease domain (PD)). Among them, Kringles are responsible for the binding of fibrinolysinogen to low molecular weight and high molecular weight ligands (i.e., lysine binding activity), causing fibrinolysinogen to transform into a more open conformation, making it easier to be activated; the protease domain (PD) is the residue Val562-Asn791, tPA and UPA specifically cut the Arg561-Val562 activation bond of fibrinolysinogen, thereby forming fibrinolysin. Therefore, the protease domain (PD) is the region that confers proteolytic activity to fibrinolysinogen.

In the present invention, "fibrinolytic enzyme" and "fibrinolytic enzyme" can be used interchangeably and have the same meaning; "fibrinolytic enzyme" and "fibrinolytic enzyme progen" can be used interchangeably and have the same meaning.

In the present application, the meaning or activity of the "lack" of fibrinolysin is that the content of fibrinolysin in the subject's body is lower than that of a normal person, low enough to affect the normal physiological function of the subject; the meaning or activity of the "absence" of fibrinolysin is that the content of fibrinolysin in the subject's body is significantly lower than that of a normal person, or even the activity or expression is extremely low, and normal physiological function can only be maintained through exogenous supply.

Those with ordinary knowledge in the art can understand that all technical solutions of the present invention for plasminogen are applicable to plasminase, and therefore, the technical solutions described in the present invention cover plasminogen and plasminase. In the circulation process, plasminogen adopts a closed inactive conformation, but when it binds to a thrombus or a cell surface, it is transformed into an active plasminase in an open conformation under the mediation of a plasminogen activator (PA). The active plasminase can further hydrolyze the fibrin clot into fibrin degradation products and D-dimers, thereby dissolving the thrombus. The PAp domain of fibrinolysin contains important determinants that maintain fibrinolysin in an inactive closed conformation, while the KR domain is able to bind to lysine residues on receptors and substrates. It is known that many enzymes can act as fibrinolysin activators, including tissue fibrinolysin activator (tPA), urokinase fibrinolysin activator (uPA), kallikrein, and coagulation factor XII (Hagemann factor).

"Fibrinolytic enzyme active fragment" refers to a fragment having the activity of binding to lysine in a substrate target sequence (lysine binding activity), or the activity of exerting a proteolytic function (proteolytic activity), or a fragment having both proteolytic activity and lysine binding activity. The technical scheme of the present invention involving fibrinolytic enzyme covers the technical scheme of replacing fibrinolytic enzyme with a fibrinolytic enzyme active fragment. In some embodiments, the fibrinolytic enzyme active fragment of the present invention comprises a serine protease domain of a fibrinolytic enzyme or is composed of a serine protease domain of a fibrinolytic enzyme. In some embodiments, the profibrinolytic enzyme active fragment of the present invention comprises sequence 14, or comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14, or consists of sequence 14, or consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14. In some embodiments, the profibrinolytic enzyme active fragment of the present invention comprises a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or a conservative substitution variant thereof, or consists of a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or a conservative substitution variant thereof. In some embodiments, the fibrinolytic enzyme described in the present invention includes a protein containing the above-mentioned fibrinolytic enzyme active fragment.

At present, the methods for measuring fibrinolysinogen and its activity in the blood include: detection of tissue fibrinolysinogen activator activity (t-PAA), detection of plasma tissue fibrinolysinogen activator antigen (t-PAAg), detection of plasma tissue fibrinolysinogen activity (plgA), detection of plasma tissue fibrinolysinogen antigen (plgAg), detection of plasma tissue fibrinolysinogen activator inhibitor activity, detection of plasma tissue fibrinolysinogen activator inhibitor antigen, and detection of plasma fibrinolysin-antifibrinolysin complex (PAP). The most commonly used detection method is the chromogenic substrate method: chain kinase (SK) and chromogenic substrate are added to the test plasma. Under the action of SK, PLG in the test plasma is converted into PLM, which acts on the chromogenic substrate and is then measured by a spectrophotometer. The increase in absorbance is proportional to the activity of fibrinolytic enzyme. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmunoassay, etc. can also be used to measure the activity of fibrinolytic enzyme in the blood.

"Orthologs" refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs. Specifically, they refer to proteins or genes evolved from the same ancestral gene in different species. The fibrinolysinogen of the present invention includes human natural fibrinolysinogen, and also includes fibrinolysinogen orthologs or orthologs derived from different species and having fibrinolysinogen activity.

"Conservative substitution variants" are variants in which a given amino acid residue is changed without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing an amino acid in the amino acid sequence of the parent protein with an amino acid of similar properties (e.g., acidic, basic, hydrophobic, etc.). Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine, or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90% being the best, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

"Isolated" fibrinolysin refers to a fibrinolysin protein that has been separated and/or recovered from its natural environment. In some embodiments, the fibrinolysin is purified (1) to a purity of greater than 90%, greater than 95%, or greater than 98% by weight as determined by the Lowry method, e.g., greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated fibrinolysin also includes fibrinolysin prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and the like.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment over the full length of the compared sequences. However, for the purposes of the present invention, percentage amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to, with, or to a given amino acid sequence B) is calculated as follows:

Multiply the fraction X/Y by 100

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the term "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be to completely or partially prevent the occurrence or onset of a disease or its symptoms, partially or completely alleviate a disease and/or its symptoms, and/or partially or completely cure a disease and/or its symptoms, including: (a) preventing the occurrence or onset of a disease in a subject, who may have a predisposition to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., hindering its formation; and (c) alleviating a disease and/or its symptoms, i.e., causing the disease and/or its symptoms to subside or disappear.

The terms "individual," "subject," and "patient" are used interchangeably herein to refer to mammals, including but not limited to mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc.

"Therapeutically effective amount" or "effective amount" refers to the amount of a component of the fibrinolytic enzyme activation pathway or its related compound (e.g., fibrinolytic enzyme) sufficient to achieve the described prevention and/or treatment of the disease when administered to a mammal or other subject for the treatment of a disease. The "therapeutically effective amount" will vary depending on the component of the fibrinolytic enzyme activation pathway or its related compound (e.g., fibrinolytic enzyme) used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, etc.

Preparation of the fibrinolysinogen of the present invention

Fibrinolysinogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the peptide is synthesized chemically, the synthesis can be carried out via liquid phase or solid phase. Solid phase peptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is a suitable method for the chemical synthesis of fibrolysinogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize fibrolysinogen. Techniques for solid phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; pages 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, small insoluble porous beads are treated with functional units on which peptide chains are constructed. After repeated cycles of coupling/deprotection, the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing a new N-terminal amine that can be attached to another amino acid. The peptide remains fixed to the solid phase and is later cleaved off.

Standard recombinant methods can be used to produce the fibrinolytic enzyme of the present invention. For example, a nucleic acid encoding the fibrinolytic enzyme is inserted into an expression vector so that it is operably linked to a regulatory sequence in the expression vector. Expression regulatory sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcriptional termination sequences. The expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting a eukaryotic host cell (e.g., COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the fibrinolytic enzyme.

Suitable expression vectors are usually replicated in the host organism as episomes or as an integrated part of the host chromosomal DNA. Typically, the expression vector contains a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to facilitate detection of those cells transformed with the desired DNA sequence from an exogenous source.

Escherichia coli is an example of a prokaryotic host cell that can be used to replicate the subject antibody encoding polynucleotide. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be generated, which usually contain expression control sequences (e.g., origins of replication) that are compatible with the host cells. In addition, there are many well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage λ. The promoter generally controls expression, optionally in the case of an operator gene sequence, and has a ribosome binding site sequence, etc., to initiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (e.g., promoters), replication origins, termination sequences, etc. as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, among others.

In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in vitro in cell culture) can also be used to express and produce the anti-Tau antibodies of the present invention (e.g., polynucleotides encoding the subject anti-Tau antibodies). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells may contain expression control sequences, such as origins of replication, promoters and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the fibrinolysinogen described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The fibrinolysinogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or more, such as free of contaminants, such as cellular debris, macromolecules other than the target product, etc.

Pharmaceutical preparations

The therapeutic formulation can be prepared by mixing a component of the fibrinolytic enzyme activation pathway or its related compound (e.g., fibrinolytic enzyme) having the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) to form a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexadecylamine chloride; benzalkonium chloride; chloride), benzathine chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; o-catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody formulations are described in WO 97/04801, which is incorporated herein by reference.

The formulations of the present invention may also contain more than one active compound as required for the specific condition to be treated, preferably those whose activities complement each other and do not have adverse effects on each other.

The lysinogen of the present invention can be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The components of the fibrinolytic enzyme activation pathway of the present invention or its related compounds (e.g., fibrinolytic enzyme) for in vivo administration must be sterile. This can be easily achieved by filtering through a sterilizing filter membrane before or after freeze drying and reconstitution.

The components of the fibrinolytic enzyme activation pathway of the present invention or related compounds (such as fibrinolytic enzyme) can be prepared into sustained-release preparations. Suitable examples of sustained-release preparations include solid hydrophobic polymer semipermeable matrices having a certain shape and containing glycoproteins, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3773919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., supra), or degradable lactic acid-hydroxyacetic acid copolymers such as Lupron. DepotTM (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-hydroxyacetic acid can release molecules continuously for more than 100 days, while some hydrogels release proteins for a shorter time. Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the mechanism of coagulation is found to be the formation of intermolecular S-S bonds through thiodisulfide exchange, stabilization can be achieved by modifying the hydroxyl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

Medication and Dosage

Administration of the pharmaceutical composition of the present invention can be achieved by different ways, for example, by nasal inhalation, nebulized inhalation, nasal or eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g. via the carotid artery), intramuscular, rectal administration.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, and the like.

Medical personnel can determine dosage scheme based on various clinical factors. As known in the medical field, the dosage of any patient depends on many factors, including the patient's body shape, body surface area, age, specific compound to be used, sex, number of times and path of application, general health and other drugs used simultaneously. The dosage range of the pharmaceutical composition of the present invention comprising profibrinolysin can be, for example, about 0.0001 to 2000 mg/kg per day, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject weight. For example, the dosage can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Doses above or below this exemplary range are also contemplated, particularly in light of the factors described above. Doses intermediate within the above ranges are also within the scope of the present invention. Subjects may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 0.01-100 mg/kg for several consecutive days. Real-time evaluation of therapeutic efficacy and safety is required during the administration of the drug of the present invention.

Products or medicine boxes

One embodiment of the present invention relates to a product or kit comprising a component of a fibrinolytic enzyme activation pathway or a related compound thereof (e.g., fibrinolytic enzyme). The product preferably includes a container, a label or a package insert. Suitable containers include bottles, vials, syringes, etc. The container can be made of various materials such as glass or plastic. The container contains a composition that is effective for treating a disease or condition of the present invention and has a sterile access port (e.g., the container can be an intravenous solution bag or vial containing a stopper that can be penetrated by a hypodermic injection needle). At least one active agent in the composition is a component of a fibrinolytic enzyme activation pathway or a related compound thereof (e.g., fibrinolytic enzyme). The label on or attached to the container indicates that the composition is used to treat the condition described in the present invention. The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In addition, the product includes a package insert with instructions for use, including, for example, instructing the user of the composition to administer the composition comprising a component of a fibrinolytic enzyme activation pathway or a related compound thereof (e.g., fibrinolytic enzyme) and other drugs for treating associated diseases to the patient.

Embodiment

The fibrinolysinogen (abbreviated as fibrinolysinogen) used in all the following examples was from human donor plasma based on the methods described in the following references: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry , 1965 , 240 (1) :541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25;251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Apr;235:1005-10, and the process was optimized and purified from human donor plasma. The fibronectin monomer was >98%.

Embodiment 1 exist PBS In the buffer system, fibronectin promotes the Aβ )degradation

Eppendorf (EP) tubes were set up as ① blank control, ② vehicle control, ③ fibronectin group, and ④ fibronectin + tPA group, with 4 tubes in each group. The blank control group was added with 43.3 μL of saline, 16 μL of lysinogen solution (0.575 mg/mL), 10 μL of ultrapure water, and 30.7 μL of PBS buffer (10 mM, pH 7.4, Thermo Fisher, 10010-031); the vehicle control group was added with 43.3 μL of Aβ40 (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011521, 1.0 mg/mL), 16 μL of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), 10 μL of ultrapure water, and 30.7 μL of PBS buffer; the lysinogen group was added with 43.3 μL Aβ40 (1.0 mg/mL), 16 μL of fibronectin solution (0.575 mg/mL), 10 μL of ultrapure water, and 30.7 μL of PBS buffer were added; 43.3 μL of Aβ40 (1.0 mg/mL), 8 μL of fibronectin solution (1.15 mg/mL), 8 μL of tPA solution (1.0 mg/mL), 10 μL of lysine solution (0.1 mM), and 30.7 μL of PBS buffer were added to the fibronectin + tPA group. After that, the cells were incubated at 37°C for 3 h, and 100 μL of 0.1% trifluoroacetic acid solution was added to terminate the reaction.

According to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320), 20% gel was prepared. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, centrifuged for 1 minute after cooling, and then 20 μL was taken for loading. The electrophoresis conditions were 30V for 1 hour, and then electrophoresed at 100V to the bottom of the gel. After electrophoresis, the stripped gel was placed in 1‰ Coomassie brilliant blue staining solution (1g Coomassie brilliant blue R250 dissolved in 1000ml ethanol: glacial acetic acid: purified water with a volume ratio of 5:2:13) for staining for 30 minutes, and then decolorized with decolorizing solution (purified water: glacial acetic acid: anhydrous ethanol = 17:2:1 volume ratio) until clean. The gel was quantitatively scanned and photographed under a gel camera.

The aggregation of amyloid protein (Aβ) is a key factor in the development of Alzheimer's disease. Among them, Aβ40 and Aβ42 containing 40 and 42 residues are deposited in the hippocampus and striatum of the brain to form senile plaques, which are the main pathogenic factors of AD ^[1] . The detection of Aβ40 and Aβ42 levels in cerebrospinal fluid has gradually become a physiological indicator of clinical Alzheimer's disease.

The results showed that the amount of Aβ40 in the vehicle control group did not change, which was defined as 100%; in the fibrinolysinogen group, Aβ40 was partially degraded when fibrinolysinogen alone was added; in the fibrinolysinogen + tPA group, Aβ40 was significantly degraded in vitro when fibrinolysinogen and tPA were added, which was significantly different from the vehicle control group (** represents P < 0.01) (Figure 1). This shows that in the PBS buffer system, fibrinolysinogen can promote the degradation of Aβ40.

Embodiment 2 In rabbit cerebrospinal fluid, fibronectin promotes the expression of amyloid protein ( Aβ )degradation

Eppendorf (EP) tubes were set up as ① blank control, ② solvent control, and ③ fibronectin group, 4 in each group. The blank control group was added with 43.3 μL of saline, 16 μL of fibronectin solution (0.575 mg/mL), and 40.7 μL of rabbit cerebrospinal fluid; the solvent control group was added with 43.3 μL of Aβ40 (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011521, 1.0 mg/mL), 16 μL of solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, PH7.4), and 40.7 μL of rabbit cerebrospinal fluid; the fibronectin group was added with 43.3 μL of Aβ40 (1.0 mg/mL), 16 μL of fibronectin solution (0.575 mg/mL), and 40.7 μL of rabbit cerebrospinal fluid. After that, the mixture was incubated at 37°C for 3 h, and then 100 μL of 0.1% trifluoroacetic acid solution was added to terminate the reaction.

According to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320), 20% gel was prepared. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, centrifuged for 1 minute after cooling, and then 20 μL was taken for loading. The electrophoresis conditions were 30V for 1 hour, and then electrophoresed at 100V to the bottom of the gel. After electrophoresis, the stripped gel was placed in 1‰ Coomassie brilliant blue staining solution (1g Coomassie brilliant blue R250 dissolved in 1000ml ethanol: glacial acetic acid: purified water with a volume ratio of 5:2:13) for staining for 30 minutes, and then decolorized with decolorizing solution (purified water: glacial acetic acid: anhydrous ethanol = 17:2:1 volume ratio) until clean. The gel was quantitatively scanned and photographed under a gel camera.

The results showed that the amount of Aβ40 in the vehicle control group did not change, which was defined as 100%; in the fibrinolysin group, when fibrinolysin was added alone, Aβ40 was partially degraded to 74.81% (Figure 2). This indicates that fibrinolysin can promote the degradation of Aβ40 in the rabbit cerebrospinal fluid.

Embodiment 3 In the brain homogenate of Alzheimer's disease model and normal mice, profibrinolytic enzyme promotes the expression of human amyloid protein Aβ40 degradation

Four B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (abbreviated as FAD) and four C57BL/6 (normal) mice aged 11 weeks were selected. After sacrifice, the whole brain tissue was obtained and weighed and placed in an Eppendorf (EP) tube. 1×PBS (Thermo Fisher, pH7.4; 10010-031) was added at a ratio of 150 mg tissue/mL PBS, and the mixture was homogenized at 4°C (1 min/time, 3-4 times). After homogenization, the mixture was centrifuged at 4°C (12000 rpm, 15 min), and the supernatant brain homogenate was placed in a new EP tube.

Eppendorf (EP) tubes were set up as ① blank control group, ② solvent control group, and ③ fibrolysin group, with 5 parallels in each group. The blank control group was added with 21.5 μL of physiological saline, 4.6 μL of fibrinolysin solution (2 mg/mL), and 23.9 μL of mouse brain homogenate; the vehicle control group was added with 21.5 μL of Aβ40 (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011521, 1.0 mg/mL), 4.6 μL of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, PH7.4), and 23.9 μL of mouse brain homogenate; the fibrinolysin group was added with 21.5 mL of Aβ40 (1.0 mg/mL), 4.6 μL of fibrinolysin solution (2 mg/mL), and 23.9 μL of mouse brain homogenate. After adding the samples of each group, incubate at 37℃ for 6h and then add 50μL 0.1% trifluoroacetic acid solution to terminate the reaction.

According to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320), 20% gel was prepared. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100℃ for 5min, centrifuged for 1min after cooling, and then 20μL was loaded. The electrophoresis conditions were 30V for 1h, and then 100V electrophoresis to the bottom of the gel. After electrophoresis, the stripped gel was placed in 1‰ Coomassie brilliant blue staining solution (1g Coomassie brilliant blue R250 dissolved in 1000ml ethanol: glacial acetic acid: purified water with a volume ratio of 5:2:13) for staining for 30 minutes, and then decolorized with decolorizing solution (purified water: glacial acetic acid: anhydrous ethanol = 17:2:1 volume ratio) until clean. The gel was photographed and quantitatively scanned under a biomolecular imaging instrument.

The results showed that in the brain plasma of Alzheimer's disease model mice, the amount of human amyloid protein Aβ40 in the fibrinolysin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** indicates P < 0.001 =; in the brain plasma of normal mice, the amount of amyloid protein Aβ40 in the fibrinolysin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (P = 0.001) (Figure 3). This shows that in the brain plasma of Alzheimer's disease model and normal mice, fibrinolysin can effectively promote the degradation of human amyloid protein Aβ40.

Embodiment 4 In the brain plasma of normal mice and Alzheimer's disease models, profibrinolytic enzymes promote the expression of human amyloid protein Aβ42 degradation

Four B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (abbreviated as FAD) and four C57BL/6 (normal) mice aged 11 weeks were selected. After sacrifice, the whole brain tissue was obtained and weighed and placed in an Eppendorf (EP) tube. 1×PBS (Thermo Fisher, pH7.4; 10010-031) was added at a ratio of 150 mg tissue/mL PBS, and the mixture was homogenized at 4°C (1 min/time, 3-4 times). After homogenization, the mixture was centrifuged at 4°C (12000 rpm, 15 min), and the supernatant brain homogenate was collected and placed in a new EP tube for later use.

Eppendorf (EP) tubes were set up as ① blank control group, ② solvent control group, and ③ fibrolysin group, with 5 parallels in each group. The blank control group was added with 21.5 μL of saline, 4.6 μL of lysozyme solution (2 mg/mL), and 23.9 μL of mouse brain homogenate; the vehicle control group was added with 21.5 μL of Aβ42 (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011526, 1.0 mg/mL), 4.6 μL of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, PH7.4), and 23.9 μL of mouse brain homogenate; the lysozyme group was added with 21.5 mL of Aβ42 (1.0 mg/mL), 4.6 μL of lysozyme solution (2 mg/mL), and 23.9 μL of mouse brain homogenate. After adding the samples of each group, incubate at 37℃ for 6h and then add 50μL 0.1% trifluoroacetic acid solution to terminate the reaction.

According to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320), 20% gel was prepared. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100℃ for 5min, centrifuged for 1min after cooling, and then 20μL was loaded. The electrophoresis conditions were 30V for 1h, and then 100V electrophoresis to the bottom of the gel. After electrophoresis, the stripped gel was placed in 1‰ Coomassie brilliant blue staining solution (1g Coomassie brilliant blue R250 dissolved in 1000ml mixed solution (ethanol: glacial acetic acid: purified water volume ratio of 5:2:13)) for staining for 30 minutes, and then decolorized with decolorizing solution (purified water: glacial acetic acid: anhydrous ethanol = 17:2:1 volume ratio) until clean. The gel was photographed and quantitatively scanned under a biomolecular imaging instrument.

The results showed that in the brain plasma of Alzheimer's disease model mice, the amount of fibrinolytic amyloid protein Aβ42 was lower than that of the solvent control group, and the amounts of its polymers a, b, and c were significantly lower than those of the solvent group, with extremely significant differences (* represents P < 0.05; *** represents P < 0.001 =; in the brain plasma of normal mice, the amount of fibrinolytic amyloid protein Aβ4 The amount of 2 in the FAD group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** represents P < 0.001 =, and the amounts of polymers a, b, and c were all lower than those in the vehicle group, and the difference was extremely significant (*** represents P < 0.001 = (Figure 4). This shows that in the homogenate of FAD and normal mouse brains, lysogen can effectively promote the degradation of human amyloid protein Aβ42 and its polymers.

Embodiment 5 Fiber Lysozyme promotes memory recovery in Alzheimer's disease mice

B6SJL-Tg (APPSwFlLon, PSEN1*M146L* L286V)6799Vas/Mmjax (FAD) (purchased from Jackson lab, strain number: 034840) mice are commonly used transgenic model mice for studying Alzheimer's disease. Twelve 12-week-old FAD female mice were selected and randomly divided into two groups according to body weight results, vehicle group and drug group, with 6 mice in each group. Six SJLB6 (Stock Number: 10012) female mice were selected as the normal control group. The mice in the drug group were injected with 1mg/0.1ml/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group were injected with the same volume of vehicle (4% arginine + 2% glycine solution) through the tail vein. The mice in the normal control group were not treated with drugs, and the drugs were given continuously for 5 days. The first day of drug administration was set as the first day, and the Y-maze spontaneous alternation reaction experiment was carried out on the sixth day. The Y-maze consists of three completely identical arms. There is a food supply device at the end of each arm. The analysis of the animal's feeding strategy, that is, the number of times, time, correct times, wrong times, routes and other parameters entering each arm can reflect the spatial memory ability of the experimental animals, which is now often used to evaluate learning and memory functions. This experiment fully utilizes the rodents' natural tendency to explore novel environments. Animals must rely on their previous memories to make the correct choice of arm to enter, which can effectively evaluate the animal's spatial working memory ability. During the experiment, the animal is placed at the end of an arm and allowed to explore freely for a few minutes. After a period of time, the animal is placed back into the maze for formal testing. The order and total number of times the animal enters each arm are recorded. When the animal enters different arms continuously (for example, 1, 2, 3 or 1, 3, 2), it is recorded as a correct alternation response. The animal is placed at the end of an arm, and the order in which the animal enters each arm is recorded within 8 minutes.

The maximum alternation is the total number of arm entries minus 2, and the percentage is then calculated = actual alternation/maximum alternation x 100%. The final value includes the actual number of alternations, the maximum number of alternations, the percentage of the two, the total distance traveled by the animal, and the total number of arm entries ^[2] .

Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset and is characterized by cognitive impairment, neurodegeneration, β-amyloid deposition, neurofibrillary tangles, and neuroinflammation ^[3] . FAD transgenic mice are a commonly used model animal for the development of AD therapeutic drugs.

Percentage of spontaneous alternation reactions

The percentage of spontaneous alternation reaction is the ratio of actual Alternation to maximum Alternation x 100%. The results showed that the percentage of spontaneous alternation reaction in the vehicle group was significantly increased compared with the normal control group; the percentage of spontaneous alternation reaction in the drug group was significantly lower than that in the vehicle control group, with statistically significant differences (* indicates P < 0.05), and close to that in the normal control group (Figure 5).

Total arm strokes

The total number of arm entries refers to the total number of arm entries of mice within a specified time. The results showed that compared with the mice in the normal control group, the total number of arm entries of the mice in the vehicle group was significantly reduced; the total number of arm entries of the mice in the drug group was significantly more than that in the vehicle control group, with statistically significant differences (* indicates P < 0.05), and close to the normal control mice (Figure 6).

Total distance traveled

The total movement distance refers to the total length of the mouse's movement track within a specified time. The results showed that compared with the normal control mice, the total movement distance of the mice in the vehicle group was significantly reduced; the total movement distance of the mice in the drug group was significantly longer than that in the vehicle control group, with statistically significant differences (* indicates P < 0.05), and was closer to the normal control mice (Figure 7).

The above results indicate that fibrolytic enzyme can promote the recovery of spontaneous alternation reaction and memory recovery in Alzheimer's model mice.

Embodiment 6 Fiber Lysozyme is reduced in the cerebral cortex of Alzheimer's model mice Aβ42 Deposition

Twenty 8-week-old male C57 mice were selected and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups, a vehicle group and a drug group, with 10 mice in each group. All mice were anesthetized and located in the granulocyte layer of the hippocampus according to the mouse stereoscopic localization atlas (based on the coordinates of the anterior halogen point: AP-2.0mm, ML ±1.5mm, DV2.0mm). Each mouse was slowly microinjected bilaterally at an injection rate of 0.5μL/min and an injection volume of 3μL. The model group mice were injected with Aβ1-42 oligomer solution to establish the Alzheimer's model ^[3] , and the model control group mice were injected with PBS solution. Preparation of Aβ1-42 oligomer solution (10μM): Take β-Amyloid (1-42) (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011521), add cold hexafluoroisopropanol, prepare the concentration to 1mg/ml, place at room temperature for 3 days, divide into 45μL/tube, i.e. 10nmol/mL, place in a fume hood overnight, place in a 25℃ drying oven to dry for 1 hour, and store at -80℃. When using, add 10μl of dimethyl sulfoxide solution to each tube for re-dissolution, add 990μL of sterile PBS solution for injection, and place at 4℃ for 24h before use. 21 days after the brain localization injection, the mice in the vehicle group and the drug group began to be administered, which was marked as day 1. The mice in the drug group were injected with 1 mg/0.1 ml/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group were injected with 0.1 ml/mouse/day of vehicle (4% arginine + 2% glycine solution) through the tail vein. The drug was administered continuously for 28 days. On the 29th day, the mice were sacrificed and the brain was fixed in 10% formaldehyde for 24-48 hours. The fixed brain tissue was paraffin-embedded after gradient dehydration with alcohol and transparent with xylene. The substantia nigra was located and sliced with a thickness of 4 μm. The slices were dewaxed and rehydrated and then washed once. The tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS, each for 5 minutes. 5% normal goat serum (Vector laboratories, Inc., USA) was sealed for 30 minutes; after the time was up, the goat serum was discarded, rabbit anti-mouse Aβ42 antibody (Abcam, ab201060) was added dropwise and incubated overnight at 4°C, and washed twice with 0.01M PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 hour, and washed twice with 0.01M PBS for 5 minutes each time. The color was developed according to the DAB reagent kit (Vector laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Dehydrated with gradient alcohol, transparentized with xylene and sealed with neutral resin, and the sections were observed under a 200x optical microscope.

The neurotoxic effect of amyloid β-protein (Aβ) plays a major role in the progression of Alzheimer’s disease ^[4] .

The above experimental results showed that the Aβ42 deposition level in the cerebral cortex of mice in the vehicle group (Figure 8A) was significantly higher than that in the drug group (Figure 8B), and the results of optical density quantitative analysis showed significant statistical differences (* indicates P < 0.05) (Figure 8C). This suggests that fibronectin can significantly reduce the Aβ42 deposition in the cerebral cortex of Alzheimer's model mice.

Embodiment 7 Fiber Lysozyme is reduced in brain tissue of Alzheimer's model mice Aβ42 level

B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799Vas/Mmjax mice (strain number: 034840, purchased from Jackson Laboratory) were backcrossed with C57BL/6J mice to produce offspring (abbreviated as B6-F1-FAD). Eighteen female B6-F1-FAD mice aged 16-17 weeks and nine female C57BL/6J mice aged 9 weeks were selected. B6-F1-FAD mice were randomly divided into two groups according to body weight and Y-maze test results, a vehicle group and a drug group, with nine mice in each group. Nine C57BL/6J mice were used as a blank control group. After the grouping was completed, the blank control group and the vehicle group were injected with the solvent (4% arginine + 2% glycine solution) through the tail vein, and the injection volume for each mouse was 5 ml/kg. The mice in the drug group were injected with fibrolysinogen through the tail vein, and the injection volume for each mouse was 50 mg/kg, and the injection was continued for 8 consecutive days. Five days after the end of the medication, 7, 7, and 6 mice in the blank group, vehicle group, and drug group were randomly sacrificed, and brain tissues were obtained. After homogenization at 4°C, the supernatant was taken, that is, the homogenate, and the total protein concentration was detected by BCA method and Western blot detection.

Prepare 16.5% gel according to the instructions of Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 100ug of total protein for loading. The electrophoresis conditions are 30V for 1.5h, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to a PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-mouse Aβ42 antibody (Abcam, ab201060) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed using Image J.

The results showed that there was a certain level of Aβ42 protein in the brain homogenate of the blank control group mice, and the Aβ42 level in the brain tissue of the vehicle group mice was significantly higher than that of the drug group mice, and the statistical P value was 0.09 (Figure 9). This suggests that fibronectin can reduce the level of Aβ42 in the brain tissue of Alzheimer's model mice.

Embodiment 8 In normal mouse brain homogenate, fibronectin promotes Tau Protein degradation

Four C57BL/6J male mice aged 11-12 weeks and weighing 18-25 g were selected. After sacrifice, the whole brain was taken out and weighed. 1×PBS (Thermo Fisher, pH7.4; 10010-031) was added at a ratio of 150 mg tissue/mL PBS, and the mixture was homogenized at 4°C (1 min, 3-4 times). After homogenization, the mixture was centrifuged at 4°C (12000 rpm, 20 min), and the supernatant, i.e. the brain homogenate, was placed in a new EP tube.

Eppendorf (EP) tubes were set up as ① blank group, ② blank control group, ③ solvent control group, and ④ tau group, with 5 replicates in each group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL tau solution (0.5 mg/mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Tau protein solution (custom-expressed human Tau protein from Nanjing GenScript Biotechnology Co., Ltd., UniProtKB - P10636-8, 1.0 mg/mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the fibronectin group; 21.5 μL of Tau protein solution (1.0 mg/mL), 4.6 μL of fibronectin solution (0.5 mg/mL), and 23.9 μL of mouse brain homogenate were added to the fibronectin group. After adding the samples of each group, incubate at 37°C for 6 hours, and then add 50 μL of 0.1% trifluoroacetic acid solution to terminate the reaction.

Prepare 10% gel according to the SDS-PAGE gel preparation instructions. Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 20μL for loading. The electrophoresis conditions are 30V for 45 min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to an activated PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit Tau protein antibody (Abcam, ab151559) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed using Image J.

Tau protein is the most abundant microtubule-associated protein. Tau protein is a phosphate-containing protein. In a normal mature brain, Tau protein molecules contain 2 to 3 phosphate groups. However, in the brains of patients with Alzheimer's disease, Tau protein is abnormally overphosphorylated, with each Tau protein molecule containing 5 to 9 phosphate groups and losing its normal biological function ^[5] .

The results showed that in the normal mouse brain homogenate, the amount of Tau protein in the fibrinolytic enzyme group was significantly lower than that in the vehicle control group, and the difference was significant (* represents P < 0.005, ** represents P < 0.01, *** represents P < 0.001) (Figure 10). This shows that fibrinolytic enzyme can promote the degradation of Tau protein in the normal mouse brain homogenate.

Embodiment 9 In the brain homogenate of Alzheimer's mice, fibronectin promotes Tau Protein degradation

Four 11-week-old B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (FAD for short) mice were selected and sacrificed, and the whole brain was removed and weighed. The brain homogenate was prepared as described in Example 8 and placed in an EP tube.

Eppendorf (EP) tubes were set up as ① blank group, ② blank control group, ③ solvent control group, and ④ lysozyme group, with 5 parallels in each group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL lysozyme solution (0.5 mg/mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Tau (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Tau protein, UniProtKB - P10636-8, 1.0 mg/mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the fibronectin group; 21.5 μL of Tau (1.0 mg/mL), 4.6 μL of fibronectin solution (0.5 mg/mL), and 23.9 μL of mouse brain homogenate were added to the fibronectin group. After adding the samples of each group, incubate at 37°C for 6 hours, and then add 50 μL of 0.1% trifluoroacetic acid solution to terminate the reaction.

Prepare 10% gel according to the SDS-PAGE gel preparation instructions. Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 20μL for loading. The electrophoresis conditions are 30V for 45 min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to an activated PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit Tau protein antibody (Abcam, ab151559) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed using Image J.

The results showed that in the homogenate of the brain of Alzheimer's mice, the amount of Tau protein in the fibrinolysin group was significantly lower than that in the vehicle control group, and the statistical difference was significant (* represents P < 0.05, ** represents P < 0.01) (Figure 11). This shows that fibrinolysin can promote the degradation of Tau protein in the homogenate of the brain of Alzheimer's mice.

Embodiment 10 Fiber Lysozyme is reduced in brain tissue of Alzheimer's model mice Tau Protein levels

B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799Vas/Mmjax mice (strain number: 034840, purchased from Jackson Laboratory) were backcrossed three times with C57BL/6J mice to produce offspring (abbreviated as B6-F3-FAD). Eighteen female B6-F3-FAD mice aged 20-25 weeks and nine female C57BL/6J mice aged 9 weeks were selected. B6-F3-FAD mice were randomly divided into two groups according to body weight and Y-maze test results, a vehicle group and a drug group, with nine mice in each group. Nine C57BL/6J mice were used as a blank control group. After the grouping was completed, the blank control group and the vehicle group were injected with the vehicle (4% arginine + 2% glycine solution) through the tail vein, and the injection volume for each mouse was 5 ml/kg. The mice in the drug group were injected with fibrolysinogen through the tail vein, and the injection volume for each mouse was 50 mg/kg, and the injection was continued for 28 days. Seven days after the end of the drug administration, mice in each group were randomly sacrificed, brain tissues were collected, and the supernatant, i.e., brain homogenate, was taken after homogenization at 4°C for total protein detection by BCA method and Western blot detection.

Prepare 10% gel according to the instructions of the SDS-PAGE gel preparation kit (Solarbio, P1320). Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 100ug of total protein for loading. The electrophoresis conditions are 30V for 1.5h, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to a PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-mouse Tau antibody (Abcam, ab151559) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed using Image J.

The results showed that the brain homogenate of the blank control group mice contained a certain level of Tau proteins of different molecular weights; the levels of Tau proteins of various molecular weights and total Tau proteins in the brain tissue of the drug-treated group mice were significantly lower than those in the vehicle group mice, and the statistical analysis of the levels of 35kd, 35-40kd, 40 kd, 54 kd molecular weight Tau proteins and total Tau protein levels in the two groups were 0.174, 0.0406, 0.052, 0.067 and 0.055, respectively (Figure 12). This indicates that fibronectin can promote the degradation of Tau proteins in the brain tissue of Alzheimer's model mice.

Embodiment 11 In the brain homogenate of Alzheimer's disease model mice, fibronectin promotes Pro-BDNF Lysis

Four 11-week-old B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (abbreviated as FAD) mice were selected and the whole brain tissue was obtained after sacrifice. The brain homogenate was prepared in an EP tube as described above.

Eppendorf (EP) tubes were set up as ① blank group, ② blank control group, ③ solvent control group, and ④ drug administration group, with 5 parallels for each group. The blank group was added with 21.5 μL of saline, 4.6 μL of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL of mouse brain homogenate; the blank control group was added with 21.5 μL of saline, 4.6 μL of pro-fibrinolytic enzyme solution (2 mg/mL), and 23.9 μL of mouse brain homogenate; the vehicle control group was added with 21.5 μL of Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom expression, UniProtKB - P23560, 1.0 mg/mL), 4.6 μL of vehicle solution (citric acid-sodium citrate solution), and 23.9 μL of mouse brain homogenate; the pro-fibrinolytic enzyme group was added with 21.5 mL Pro-BDNF (1.0 mg/mL), 4.6 μL fibronectin solution (2 mg/mL), and 23.9 μL mouse brain homogenate. After adding each group of samples, incubate at 37°C for 6 hours, and then add 50 μL 0.1% trifluoroacetic acid solution to terminate the reaction.

Prepare 12% gel according to the instructions for SDS-PAGE gel preparation. Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 20μL for loading. The electrophoresis conditions are 30V for 45min, and then electrophoresis at 100V to the bottom of the gel. After the electrophoresis, the gel is stripped and stained with 1‰ Coomassie brilliant blue staining solution (1g Coomassie brilliant blue R250 dissolved in 1000ml ethanol: glacial acetic acid: purified water with a volume ratio of 5:2:13) for 30min, and then decolorized with decolorizing solution (purified water: glacial acetic acid: anhydrous ethanol = 17:2:1 volume ratio mixture) until clean. The gel was photographed and quantitatively scanned and analyzed under a biomolecular imaging instrument.

Brain-derived neurotrophic factor (BDNF) is an alkaline protein with a molecular weight of 12.3 kD. It is composed of 119 amino acid residues and contains three pairs of disulfide bonds. It exists in the body as a dimer and is synthesized in the form of BDNF precursor. BDNF precursor (Pro-BDNF) can be cleaved by enzymatic hydrolysis to form mature BDNF. Literature reports that Pro-BDNF has opposite effects to the mature BDNF formed by its cleavage. Pro-BDNF promotes neuronal apoptosis and reduces synaptic plasticity [6]. Mature BDNF and its receptors are widely distributed in the central nervous system. During the development of the central nervous system, they play an important role in the survival, differentiation, growth and development of neurons. They can also prevent neurons from being damaged and dying, improve the pathological state of neurons, and promote the regeneration and differentiation of damaged neurons. They are also necessary for the survival and normal physiological functions of mature neurons in the central and peripheral nervous systems ^[7] .

The results showed that in the brain homogenate of Alzheimer's disease model mice, the amount of Pro-BDNF in the fibrinolysin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (* represents P < 0.05, *** represents P < 0.001) (Figure 13). This suggests that fibrinolysin can promote the cleavage of Pro-BDNF in the brain homogenate of Alzheimer's disease model mice.

Embodiment 12 In the brain homogenate of Alzheimer's disease model mice, fibronectin promotes Pro-BDNF Cleavage to form mature BDNF

Four 11-week-old B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (abbreviated as FAD) mice were selected and the whole brain tissue was obtained after sacrifice. The brain homogenate was prepared in an EP tube as described above.

Eppendorf (EP) tubes were set up as ① blank group, ② blank control group, ③ solvent control group, and ④ drug administration group, with 5 parallels for each group. The blank group was added with 21.5 μL of saline, 4.6 μL of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL of mouse brain homogenate; the blank control group was added with 21.5 μL of saline, 4.6 μL of pro-fibrinolytic enzyme solution (2 mg/mL), and 23.9 μL of mouse brain homogenate; the vehicle control group was added with 21.5 μL of Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom expression, UniProtKB - P23560, 1.0 mg/mL), 4.6 μL of vehicle solution (citric acid-sodium citrate solution), and 23.9 μL of mouse brain homogenate; the pro-fibrinolytic enzyme group was added with 21.5 mL Pro-BDNF (1.0 mg/mL), 4.6 μL fibronectin solution (2 mg/mL), and 23.9 μL mouse brain homogenate. After adding each group of samples, incubate at 37°C for 6 hours, and then add 50 μL 0.1% trifluoroacetic acid solution to terminate the reaction.

Prepare 12% gel according to the SDS-PAGE gel preparation instructions. Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 20μL for loading. The electrophoresis conditions are 30V for 45min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to a PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-human BDNF antibody (Boster Biological Technology, PB9075) was added and incubated at room temperature for 3 h. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 h. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and the band optical density was quantitatively analyzed using Image J.

The results showed that in the brain homogenate of Alzheimer's disease model mice, the amount of Pro-BDNF in the fibroblast group was significantly lower than that in the vehicle control group, and the difference was extremely significant (** represents P < 0.01, *** represents P < 0.001); the amount of BDNF in the fibroblast group was significantly higher than that in the vehicle control group, and the difference was extremely significant (Figure 14). This suggests that fibroblast can promote the cleavage of Pro-BDNF and the formation of mature BDNF in the brain homogenate of Alzheimer's disease model mice.

Embodiment 13 Fiber Lysozyme in the hippocampus of Alzheimer's disease model mice BDNF Expression

Twenty-three 24-week-old male C57 mice were taken and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups: a blank control group of 7 mice and a model group of 16 mice. All mice were anesthetized and the Alzheimer's model was established as described in Example 6 ^[3] . 28 days after brain localization injection, all mice were weighed and tested in the Y-maze. Abnormal mice in the blank control group and model group were excluded based on the test results. The model group mice were randomly divided into two groups: a vehicle group of 6 mice, a drug group of 7 mice, and a blank control group of 6 mice. The mice in the vehicle group and the drug group began to be treated with drugs, which was marked as the first day. The mice in the drug group were injected with 1mg/0.1ml/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group were injected with 0.1ml/mouse/day of vehicle (4% arginine + 2% glycine solution) through the tail vein. The drugs were continuously administered for 28 days, and the mice in the blank control group were not treated with drugs. On the 29th day, the mice were sacrificed and the brains were fixed in 10% formaldehyde for 24-48 hours. The fixed brain tissues were paraffin-embedded after gradient dehydration with alcohol and transparent with xylene. The substantia nigra was located and sliced with a thickness of 4μm. The slices were dewaxed and rehydrated and then washed once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS, each for 5 minutes. 5% normal goat serum (Vector laboratories, Inc., USA) was sealed for 30 minutes; after the time was up, the goat serum was discarded, rabbit anti-mouse BDNF antibody (BosterBio, PB9075) was added and incubated overnight at 4°C, and washed twice with 0.01M PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 hour, and washed twice with 0.01M PBS for 5 minutes each time. The color was developed according to the DAB reagent kit (Vector laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Dehydrated with gradient alcohol, transparent with xylene and sealed with neutral resin, and the sections were observed under a 200x optical microscope.

The results showed that the blank control group (Figure 15A) mice hippocampus expressed a certain level of BDNF (indicated by arrows), the vehicle group (Figure 15B) mice hippocampus BDNF expression was significantly lower than the blank control group, and the drug group (Figure 15C) mice hippocampus BDNF expression was significantly higher than the vehicle group, and the statistical difference was significant (* indicates P < 0.05) (Figure 15D). This shows that fibronectin can promote the expression of BDNF in the hippocampus of Alzheimer's model mice.

Embodiment 14 In the brain homogenate of Alzheimer's disease model mice, fibronectin promotes Pro-NGF Cleavage to form mature NGF

Four 11-week-old B6SJLTg (APPSwFlLon, PSEN1*M146L*L286V) 6799 Vas/ Mmjax (FAD) (Stock Number: 034840) (abbreviated as FAD) mice were selected and the whole brain tissue was obtained after sacrifice. The brain homogenate was prepared in an EP tube as described above.

Eppendorf (EP) tubes were set up as ① blank control group, ② blank group, ③ vehicle control group, and ④ fibrolysinogen group, with 5 parallels in each group. The blank control group was added with 21.5 μL saline, 4.6 μL vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank group was added with 21.5 μL saline, 4.6 μL fibrinolysin solution (2 mg/mL), and 23.9 μL mouse brain homogenate; the vehicle control group was added with 21.5 μL Pro-NGF solution (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Pro-NGF, sequence source UniProtKB - P01138, 1.0 mg/mL), 4.6 μL vehicle solution, and 23.9 μL mouse brain homogenate; the fibrinolysin group was added with 21. Pro-NGF solution (1.0 mg/mL), 4.6 μL fibronectin solution (2 mg/mL), and 23.9 μL mouse brain homogenate. After adding each group of samples, incubate at 37°C for 6 hours, and then add 50 μL 0.1% trifluoroacetic acid solution to terminate the reaction.

Prepare 15% gel according to the SDS-PAGE gel preparation instructions. Mix each group of samples with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heat at 100℃ for 5 min, centrifuge for 2 min after cooling, and then take 20μL for loading. The electrophoresis conditions are 30V for 30min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel is stripped and transferred to an activated PVDF membrane (GE, A29433753) at 15V for 2.5h. After transfer, the PVDF membrane was immersed in blocking solution (5% degreased emulsion) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-human NGF antibody (Abcam, ab52918) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and the band optical density was quantitatively analyzed using Image J.

Nerve growth factor (NGF) is an important member of the neurotrophic factor family. It is synthesized in vivo in the form of precursors, including signal peptides, leader peptides, and mature peptides. Studies have reported that the precursor of NGF (Pro-NGF) and its cleavage-formed NGF have opposite effects. Pro-NGF can promote neuronal apoptosis. Mature NGF participates in regulating the growth, development, differentiation, survival, and repair of nerve cells after injury, and also plays an important regulatory role in the functional expression of central and peripheral neurons ^[8] .

The results showed that in the brain homogenate of Alzheimer's disease model mice, the amount of Pro-NGF in the fibroblast group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** indicates P < 0.001); the amount of NGF in the fibroblast group was significantly higher than that in the vehicle control group, and the difference was significant (Figure 16). This suggests that fibroblast can promote the cleavage of Pro-NGF and the formation of mature NGF in the brain homogenate of Alzheimer's disease model mice.

Embodiment 15 Fiber Lysozyme promotes the recovery of anxiety and depression in Alzheimer's model mice

Twenty-eight male C57 mice were taken and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups: a blank control group of 8 mice and a model group of 20 mice. After grouping, the Alzheimer's model was established as described in Example 6 ^[3] . 65 days after brain localization injection, all mice were tested in a water maze. The model control group was the blank control group mice. According to the test results, the abnormal mice in the blank control group and the model group were excluded. The model group mice were randomly divided into two groups: a vehicle group of 10 mice, a drug group of 10 mice, and a blank control group of 8 mice. After the grouping was completed, the first phase of drug administration began for the mice in the vehicle group and the drug administration group: the mice in the drug administration group were injected with 50 mg/kg/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group and the blank control group were injected with 5 ml/kg/mouse/day of vehicle (4% arginine + 2% glycine solution) through the tail vein, and the drug administration was continued for 28 days. The second phase of drug administration began 50 days after the end of the first phase of drug administration, and the drug administration method was the same as the first phase, and the drug administration was continued for 7 days. The field experiment was carried out on the 8th day of the second phase of drug administration.

Field experiment

During the experiment, the mouse was placed in the center of the bottom of the open field (40x40x40cm), and video and timing were performed simultaneously. The observation lasted for 5 minutes, and each mouse was subjected to 3 experiments. The Smart system is a complete and easy-to-use video tracking system for evaluating the behavior of experimental animals. It allows recording of tracks, activities, specific behaviors (such as rotation, stretching and rearing) and events, and performs calculations of various analysis parameters. This experiment used the Smart3.0 system to record and analyze the movement of mice. Parameters included the total movement distance in the border zone and the movement distance in the center zone. In each experiment, 70% alcohol was used to wipe the box to prevent olfactory preference ^[1] .

The design principle of the open field experiment is based on the avoidance of mice, which means that mice are afraid of open, unknown, and potentially dangerous places, so they have the nature of "sticking to the wall" activities. The total distance and average speed are regarded as the main data reflecting the spontaneous activities of mice. Avoidance is evaluated by the activities of mice in the peripheral areas (four corners and four sides) of the open field. From the perspective of the activity time in the peripheral area reflecting avoidance, the time is reduced, indicating that the mice are more "adventurous". The activity time in the central area is significantly increased, indicating that the avoidance and anxiety (depression) levels are lower.

Percentage of distance traveled in the border zone

The percentage of movement distance in the border zone is the ratio of the length of the mouse's movement track in the border zone to the total movement track length within a specified time. The results showed that the blank control group had a certain percentage of movement distance in the border zone, the percentage of movement distance in the border zone of the mice in the vehicle group was significantly higher than that in the blank control group, and the percentage of movement distance in the border zone of the mice in the drug group was significantly lower than that in the vehicle group, and the statistical difference was close to significant (P-0.08) (Figure 17). This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Central area movement distance percentage

The percentage of central area movement distance is the ratio of the length of the mouse's movement track in the central area to the total movement track length within a specified time. The results showed that the blank control group had a certain percentage of central area movement distance, the percentage of central area movement distance of the vehicle group mice was significantly smaller than that of the blank control group, and the percentage of central area movement distance of the drug group mice was significantly greater than that of the vehicle group, and the statistical difference was close to significant (P-0.08) (Figure 18). This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Embodiment 16 Fiber Lysozyme promotes the recovery of anxiety and depression in Alzheimer's model mice

Twenty-eight male C57 mice were taken and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups: a blank control group of 8 mice and a model group of 20 mice. After grouping, the Alzheimer's model was established as described in Example 6 ^[3] . 65 days after brain localization injection, all mice were tested in a water maze. The model control group was the blank control group mice. According to the test results, the abnormal mice in the blank control group and the model group were excluded. The model group mice were randomly divided into two groups: a vehicle group of 10 mice, a drug group of 10 mice, and a blank control group of 8 mice. After the grouping was completed, the first phase of drug administration began for the mice in the vehicle group and the drug administration group: the mice in the drug administration group were injected with 50 mg/kg/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group and the blank control group were injected with 5 ml/kg/mouse/day of vehicle (4% arginine + 2% glycine solution) through the tail vein, and the drug administration was continued for 28 days. The second phase of drug administration began 50 days after the end of the first phase of drug administration, and the drug administration method was the same as the first phase, and the drug administration was continued for 9 days. Two days after the end of the second phase of drug administration, the elevated plus maze behavior test was performed.

The elevated cross maze uses the contradictory and conflicting behaviors of animals' exploration characteristics of new and strange environments and their fear of high-hanging open arms to examine the anxiety state of animals. The elevated cross maze has a pair of open arms and a pair of closed arms. Rods tend to move in the closed arms due to their dark preference, but they will move in the open arms due to their curiosity and exploratory nature. When faced with novel stimuli, animals simultaneously have the impulse to explore and fear, which creates conflicting behaviors of exploration and avoidance, thereby generating anxiety. Anti-anxiety drugs can significantly increase the number and time of entering the open arms. The cross maze is higher than the ground, which is equivalent to a person standing on a cliff, causing the experimental subjects to have fear and anxiety. The elevated plus maze is widely used in scientific research and computer-assisted teaching in many disciplines such as new drug development/screening/evaluation, pharmacology, toxicology, preventive medicine, neurobiology, animal psychology and behavioral biology. It is a classic experiment for medical schools and scientific research institutions to conduct behavioral research, especially anxiety and depression research.

At the beginning of the experiment, the mice were placed in the maze from the central grid to the closed arm, and their activities were recorded within 5 minutes. The observation indicators included: the number of open arm entries (two front claws must enter the arm), the open arm stay time, the number of closed arm entries, and the closed arm stay time. The proportion of open arm stay time, the proportion of open arm entries, and the total number of entries in the elevated plus maze were calculated. After the experiment was completed, the mice were taken out, the two arms were cleaned, and alcohol was sprayed to remove the odor. Finally, the data were analyzed using animal behavior software.

Total distance traveled

The total movement distance refers to the total movement track length of mice within the specified recording time. The results showed that the blank control group had a certain total movement distance; the movement distance of the vehicle group was significantly greater than that of the blank control group; the total movement distance of the drug group was significantly less than that of the vehicle group, and the statistical difference was extremely significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 19), and was closer to the total movement distance of mice in the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Closing arm Sports course

The closed arm movement distance refers to the length of the closed arm movement track within a specified time. The results showed that the mice in the blank control group had a certain closed arm movement distance; the closed arm movement distance of the vehicle group was significantly greater than that of the blank control group; the closed arm movement distance of the drug group was significantly less than that of the vehicle group, and the statistical difference between the two groups was significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 20), and the closed arm movement distance of the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Closing arm Movement distance percentage

The percentage of closed arm movement distance refers to the ratio of the closed arm movement track length to the total movement track length within a specified time. The results showed that the blank control group mice had a certain percentage of closed arm movement distance; the percentage of closed arm movement distance in the vehicle group was significantly greater than that in the blank control group; the percentage of closed arm movement distance in the drug group was significantly less than that in the vehicle group, and the statistical difference between the two groups was significant (* indicates P < 0.05) (Figure 21), and the percentage of closed arm movement distance in the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Closing arm Number of entries

The results showed that the mice in the blank control group had a certain number of closed arm entries; the number of closed arm entries in the vehicle group was significantly higher than that in the blank control group; the number of closed arm entries in the drug group was significantly lower than that in the vehicle group, and the statistical difference between the two groups was extremely significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 22), and the number of closed arm entries in the drug group was closer to that in the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behaviors in Alzheimer's model mice.

Closing arm time

The closed arm time refers to the time that mice can stay in the closed arm for a specified time. The results showed that the mice in the blank control group had a certain closed arm time, the closed arm time of the mice in the vehicle group was significantly shorter than that of the blank control group, and the closed arm time of the mice in the drug group was significantly longer than that of the vehicle group. The statistical difference between the two groups was significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 23), and the closed arm time of the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Closing arm Percentage of time

The percentage of closed arm time refers to the ratio of the time the mouse stays in the closed arm to the total recorded time. The results showed that the blank control group mice had a certain percentage of closed arm time, the percentage of closed arm time of the vehicle group mice was significantly lower than that of the blank control group, and the percentage of closed arm time of the drug group mice was significantly higher than that of the vehicle group. The statistical difference between the two groups was significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 24), and the percentage of closed arm time of the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Closed arm average speed

The average speed of the closed arm refers to the ratio of the movement distance of the closed arm to the time of the closed arm. The results showed that the blank control group had a certain average movement speed of the closed arm, the average movement speed of the closed arm of the vehicle group was significantly greater than that of the blank control group, and the average movement speed of the closed arm of the drug group was significantly less than that of the vehicle group. The statistical difference between the two groups was extremely significant (** indicates P < 0.01) (Figure 25), and the average movement speed of the closed arm of the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depression behavior in Alzheimer's model mice.

Embodiment 17 Fiber Lysozyme promotes memory recovery in Alzheimer's disease mice

Twenty-eight male C57 mice were taken and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups: a blank control group of 8 mice and a model group of 20 mice. After grouping, the Alzheimer's model was established as described in Example 6 ^[3] . 65 days after brain localization injection, all mice were tested in a water maze. The model control group was the blank control group mice. According to the test results, the abnormal mice in the blank control group and the model group were excluded. The model group mice were randomly divided into two groups: a vehicle group of 10 mice, a drug group of 10 mice, and a blank control group of 8 mice. After the grouping was completed, the first phase of drug administration began for the mice in the vehicle group and the drug administration group: the mice in the drug administration group were injected with 50 mg/kg/mouse/day of fibrolysinogen through the tail vein, and the mice in the vehicle group and the blank control group were injected with 5 ml/kg/mouse/day of vehicle (4% arginine + 2% glycine solution) through the tail vein, and the drug administration was continued for 28 days. The second phase of drug administration began 50 days after the end of the first phase of drug administration, and the drug administration method was the same as the first phase, and the drug administration was continued for 9 days. Two days after the end of the second phase of drug administration, Y maze behavior testing was performed.

The results showed that the percentage of spontaneous alternation reaction in the vehicle group was significantly lower than that in the vehicle group, and the percentage of spontaneous alternation reaction in the drug group was significantly higher than that in the vehicle group. The statistical difference between the two groups was significant (* indicates P < 0.05), and the drug group was close to the blank control mice (Figure 26). This suggests that fibronectin can promote the recovery of memory function in Alzheimer's model mice.

Embodiment 18 Fiber Lysozyme promotes the recovery of anxiety and depression in Alzheimer's model mice

Eighteen B6SJL-Tg (APPSwFlLon, PSEN1*M146L* L286V)6799Vas/Mmjax (breeder mice purchased from Jackson lab, strain number: 034840) female mice aged 20-25 were selected and randomly divided into two groups according to body weight and Y-maze test results, 9 mice in the vehicle group and 9 mice in the drug group. Nine C57 female mice of the same age group were first selected as the blank control group. After the grouping was completed, the blank control group and the vehicle group mice were injected with the solvent (4% arginine + 2% glycine solution) through the tail vein at an injection volume of 5ml/kg. The drug group mice were intravenously injected with fibrolysinogen at an injection dose of 50mg/kg for 18 consecutive days. On the 19th day, the elevated plus maze behavioral experiment was conducted.

The results showed that the mice in the blank control group had a certain distance of closed arm movement; the closed arm movement distance of the mice in the vehicle group was significantly less than that of the mice in the blank control group; the closed arm movement distance of the mice in the drug group was significantly greater than that of the vehicle group, and the statistical difference between the two groups was significant (** indicates P < 0.01, *** indicates P < 0.001) (Figure 27), and the closed arm movement distance of the drug group was closer to the blank control group. This suggests that fibronectin can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Embodiment 19 Fiber Lysozyme improves hippocampal damage in Alzheimer's disease mice

B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799Vas/Mmjax mice (strain number: 034840, purchased from Jackson Laboratory) were backcrossed with C57BL/6J mice to produce offspring (abbreviated as B6-F1-FAD). Eighteen female B6-F1-FAD mice aged 16-17 weeks and nine female C57BL/6J mice aged 9 weeks were selected. B6-F1-FAD mice were randomly divided into two groups according to body weight and Y-maze test results, a vehicle group and a drug group, with nine mice in each group. Nine C57BL/6J mice were used as a blank control group. After the grouping was completed, the blank control group and the vehicle group were injected with the solvent (4% arginine + 2% glycine solution) through the tail vein, and the injection volume for each mouse was 5ml/kg. The mice in the drug group were injected with 50mg/kg of fibronectin through the tail vein, and the injection volume for each mouse was 8 consecutive days. Five days after the end of the drug administration, the mice were sacrificed, and the brain tissue was fixed in 10% neutral formaldehyde solution for 24-48 hours. The fixed brain tissue was paraffin-embedded after alcohol gradient dehydration and xylene transparency. The slice thickness was 3μm, the slice was dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol differentiation, ammonia blue, alcohol gradient dehydration and sealing, and the slice was located and observed under a 200x optical microscope for hippocampal status.

The results showed that the morphology of the hippocampus tissue of the blank control group (Figure 28A) mice was normal; compared with the vehicle group (Figure 28B), the morphology of the hippocampus tissue of the drug group (Figure 28C) was significantly improved. This suggests that fibronectin can improve hippocampal damage in Alzheimer's model mice.

Embodiment 20 Effects of Fibrolysinogen on Alzheimer's Disease Patients Who Voluntarily Undergo Therapy

The following patients signed informed consent, voluntarily took the medication, and obtained approval from the hospital ethics committee.

Patient 1 , male, 76 years old, had memory loss six months ago, his personality became stubborn, and his memory and learning abilities declined. He was clinically diagnosed with Alzheimer's disease. Medication regimen: 50 mg intravenous injection, add 10 mg per day, and start atomization inhalation of 10 mg from the third day. Take the medicine once a day for 13 days.

Efficacy: Family members said that during the course of medication, the patient's mental state gradually improved, his reaction sensitivity gradually and significantly improved, and in the later stage of medication, the patient's ability to learn new things improved and his memory improved. After 13 days of medication, the overall physical feeling improved by about 50%, and memory improved by about 50%.

It shows that fibrolytic enzyme can improve the memory, learning ability and mental state of Alzheimer's patients.

Patient 2 , female, 96 years old. Medical history: hypertension for more than 20 years, no history of heart disease and diabetes. About half a year ago, the patient developed memory loss, learning ability deterioration, thinking ability and communication ability decline, irritability, emotional indifference, and the Mini-Mental State Examination (MMSE) test score was 10 points.

Dosage plan: Nebulization combined with intravenous injection. Day 1: Nebulization inhalation 3 times, 5 mg each time, starting from the second day, nebulization as before, starting from 30 mg, intravenous injection, add 10 mg every day, and continue medication for 7 days.

The Mini-Mental State Examination (MMSE) can comprehensively, accurately and quickly reflect the intellectual status and cognitive impairment of the subjects. This scale is simple and easy to use, widely used at home and abroad, and is the preferred scale for dementia screening. Scoring reference: 27-30 points, normal; score <27 points, cognitive impairment; score 21-26, mild; score 10-20, moderate; score 0-9, severe.

Efficacy: 1. Mental state improved; 2. Elderly dementia symptoms improved, memory improved; 3. Elderly anxiety reduced; 4. MMSE score 14 points, mainly reflected in the ability to follow other people's instructions. MMSE scores before and after medication are shown in Table 1.

It shows that fibrolytic enzyme can improve the MMSE score of Alzheimer's patients and improve patients' memory, thinking ability, anxiety and mental state.

Table 1 MMSE scores before and after medication MMSE score Before medication 10 After medication 14

Patient 3 , female, 87 years old, had poor memory and comprehension. She was admitted to the hospital for examination and the doctor diagnosed her with cerebral infarction. Her memory and comprehension were affected, and she showed signs of senile dementia. She was slightly confused and in a state of suberoticism. She could not correctly identify the surrounding environment, had confusion about time and place, and had difficulty communicating with others. Her overall condition was rated 10 points. (The overall condition was rated as 10 on the first day without spraying, and 10 on yesterday after spraying, which was the most serious, 1 was the lightest, and 0 was normal).

Dosage plan: 150-250 mg intravenous injection, plus nebulization inhalation 10 mg/time, once every 4 hours, 3 times a day, for 14 days. After stopping the drug for one week, continue to use the drug for one week, the method is the same as before, the nebulization inhalation dose is 15 mg/time, once every 4 hours, 3 times a day. After stopping the drug for one week, continue to use it for two weeks, every other day, 250 mg intravenous injection, nebulization 15 mg/time, once every 4 hours, 3 times a day. Later, it was changed to twice a week, the dosage was 400 mg intravenous injection, plus nebulization 10 mg/time, twice a day, and the medication was used for 2 weeks. Now it is used once a week, 500 mg intravenous injection, plus nebulization 10 mg/time, twice a day, and 2 days a week.

During the treatment, the above symptoms gradually improved. After 14 days of medication, the patient's mood improved, communication with others was basically smooth, memory was restored, and the concept of time gradually became clear. The overall condition was rated 4 points. When communicating with others, 70%-80% could be understood and answered accurately. Although the names of people were often mixed up, there was no problem with the specific orientation of the characters.

It shows that fibrolytic enzyme can improve Alzheimer's disease, including improving patients' memory, communication ability, cognitive ability, and orientation ability.

Patient 4 , female , 91 years old, was diagnosed with mild encephalopathy and mild cognitive impairment. Before medication, the patient's memory score was 10, calculation score was 10, and orientation score was 10 (the overall condition score was 10 on the first day without medication, and 10 was the most severe on the first day after medication, 1 was the lightest, and 0 was normal).

Dosage regimen: 50-100 mg intravenous injection, once a day, plus 10 mg/time by nebulization inhalation, 2-3 times a day. Take the above medication once every two days for 13 consecutive days.

After the 13th day of treatment, the patient rated his memory as 9, his calculation ability as 9, and his orientation as 9.

It shows that fibrolytic enzyme can improve patients' cognitive impairment, memory ability, calculation ability and orientation ability.

Patient 5 , female, 79 years old, had symptoms such as poor memory and bad temper 4 years ago, which gradually worsened. Now the patient is relatively quiet, can't concentrate for more than 2 minutes, has language expression disorder, and short-term memory loss. Unable to distinguish time and place, easily sad, and lost the ability to take care of herself. MMSE score is 3 points.

Dosage regimen: 50 mg intravenously, add 50 mg every two days for 14 consecutive days, then take 400 mg twice a week for a total of 30 days.

Starting from the 4th day of medication, attention and comprehension began to improve slightly; on the 7th day of medication, comprehension and attention improved further, and he was able to understand questions and try to answer them; on the 10th day of medication, he was able to recognize and remember more things and relatives; on the 14th day of medication, attention could be focused for 7 to 8 minutes; if the condition was good, the memory of what had just happened could last for 6 to 7 minutes; on the 21st day of medication, attention could be focused for more than 30 minutes; 24 days after medication, the memory of what had just happened could last for about 1 hour, and the language expression was also richer, with an MMSE score of 9 points; 30 days after medication, the MMSE score was 9 points; one month after stopping the medication, arithmetic ability improved, with an MMSE score of 8 points. The MMSE scores before and after medication are shown in Table 2

It shows that fibrolytic enzyme can improve the MMSE score of Alzheimer's patients and improve their memory, cognitive ability, attention, comprehension, language expression and calculation ability.

Table 2 MMSE scores before and after medication MMSE score Before medication 3 After medication (24 days) 9 After medication (30 days) 9 After stopping medication (30 days) 8

References

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[4]Park S W , Kim J H , Mook-Jung I , et al. Intracellular amyloid beta alters he tight junction of retinal pigment epithelium in 5XFAD mice[J]. Neurobiology of Aging, 2014, 35(9):2013-2020.

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[8]Aloe L, Rocco M L, Bianchi P, et al. Nerve growth factor: from the early discoveries to the potential clinical use[J]. Journal of Translational Medicine, 2012, 10(1).

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Figure 1A-B shows the Tricine-SDS electrophoresis results of amyloid protein Aβ40 solubilized by fibrinolysin in a PBS buffer system. A is Tricine-SDS-PAGE, and B is the quantitative scanning analysis results of Aβ40 dissolution in vitro. The results showed that the amount of Aβ40 in the vehicle control group was defined as 100%, and there was no change; in the fibrinolysin group, when fibrinolysin was added alone, Aβ40 was partially degraded; in the fibrinolysin+tPA group, when fibrinolysin and tPA were added, Aβ40 was significantly degraded in vitro, which was significantly different from the vehicle control group (** represents P < 0.01). This shows that fibrinolysin can promote the degradation of Aβ40.

Figure 2A-B shows the Tricine-SDS electrophoresis results of amyloid protein Aβ40 solubilized by fibrinolysin in rabbit cerebrospinal fluid. Lanes 1 and 4 are blank control groups; Lane 2 is the vehicle group; Lane 3 is the fibrinolysin group. A is Tricine-SDS-PAGE, and B is the quantitative scanning analysis results of Aβ40 dissolution. The results show that the amount of Aβ40 in the vehicle control group is defined as 100%, and there is no change; in the fibrinolysin group, when fibrinolysin is added alone, Aβ40 is partially degraded to 74.81%. This shows that fibrinolysin can promote the degradation of Aβ40.

Figure 3 A-B shows the effect of fibronectin on human amyloid protein Aβ40 in rabbit cerebrospinal fluid. A is the electrophoresis of Tricine-SDS-PAGE, and B is the quantitative scanning analysis result of Aβ40 dissolution. The results show that the amount of Aβ40 in the solvent control group was defined as 100%, and there was no change; when fibronectin was added alone in the fibronectin group, Aβ40 was partially degraded to 74.81%. This shows that fibronectin can promote the degradation of human amyloid protein Aβ40 in rabbit cerebrospinal fluid.

Figure 4 A-B The effect of profibrinolytic enzyme on human amyloid protein Aβ40 in the brain homogenate of Alzheimer's disease model and normal mice. A is the Tricine-SDS-PAGE electrophoresis, and B is the quantitative scanning analysis result of Aβ40 dissolution in vitro. The results showed that in the brain homogenate of FAD mice, the amount of amyloid protein Aβ40 in the drug-treated group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** indicates P < 0.001); in the brain homogenate of normal mice, the amount of human amyloid protein Aβ40 in the drug-treated group was significantly lower than that in the vehicle control group, and the difference was extremely significant (P = 0.001). It shows that in the brain plasma of Alzheimer's disease models and normal mice, fibrolytic enzyme can effectively promote the degradation of human amyloid protein Aβ40.

Figure 5 Statistical results of the percentage of spontaneous alternation reactions in Alzheimer's model mice 5 days after administration of fibrinolysinogen. The results showed that the percentage of spontaneous alternation reactions in the vehicle group was significantly increased compared with that in the normal control mice; the percentage of spontaneous alternation reactions in the drug group was significantly lower than that in the vehicle control group, with statistically significant differences (* indicates P < 0.05), and was closer to that in the normal control mice.

Figure 6 Statistical results of total arm-entry times of Alzheimer's model mice 5 days after administration of fibrinolysinogen. The results showed that the total arm-entry times of mice in the vehicle group were significantly reduced compared with those in the normal control group; the total arm-entry times of mice in the drug-generated group were significantly higher than those in the vehicle control group, with statistically significant differences (* indicates P < 0.05), and were closer to normal control mice.

Figure 7 Statistics of the total movement distance of Alzheimer's model mice after 5 days of administration of fibrinolysinogen. The results showed that the total movement distance of mice in the vehicle group was significantly reduced compared with that of the normal control mice; the total movement distance of mice in the fibrinolysinogen group was significantly longer than that of the vehicle control group, with statistically significant differences (* indicates P < 0.05), and was closer to that of normal control mice.

Figure 8A-C Quantitative analysis of Aβ42 staining in the cerebral cortex of Alzheimer's model mice 28 days after administration of fibrinolysinogen. A is the vehicle group, B is the drug group, and C is the average optical density quantitative analysis result. The results showed that the Aβ42 deposition level in the cerebral cortex of the vehicle group mice was significantly higher than that in the drug group, and the optical density quantitative analysis results were statistically significantly different (* indicates P < 0.05). This suggests that fibrinolysinogen can reduce the deposition of Aβ42 in the cerebral cortex of Alzheimer's model mice.

Figure 9A-B Western blot results of Aβ42 in the homogenate of hindbrain tissue of Alzheimer's model mice given fibronectin for 8 days. A is a representative picture of Western blot, and B is the result of optical density quantitative analysis. The results showed that there was a certain level of Aβ42 protein in the brain homogenate of the blank control group mice, and the level of Aβ42 in the brain tissue of the vehicle group mice was significantly higher than that of the drug group mice, and the statistical P value was 0.09. This shows that fibronectin can reduce the level of Aβ42 in the brain tissue of Alzheimer's model mice.

Figure 10A-B Effects of Fibrinolysin on Tau protein in normal mouse brain homogenate. A is a Western blot image, and B is the result of quantitative analysis of the optical density of Tau protein bands. The results showed that in the normal mouse brain homogenate, the amount of Tau protein in the Fibrinolysin group was significantly lower than that in the vehicle control group, and the difference was significant (* represents P < 005, ** represents P < 0.01, *** represents P < 0.001). This suggests that Fibrinolysin can promote the degradation of Tau protein in normal mouse brain homogenate.

Figure 11A-B Effects of Fibrinolysin on Tau protein in brain homogenates of Alzheimer's mice. A is a Western blot image, and B is the result of quantitative analysis of the optical density of Tau protein bands. The results showed that in the brain homogenates of Alzheimer's mice, the amount of Tau protein in the Fibrinolysin group was significantly lower than that in the vehicle control group, and the statistical difference was significant (* represents P < 005, ** represents P < 0.01). This suggests that Fibrinolysin can promote the degradation of Tau protein in the brain homogenates of Alzheimer's mice.

Figure 12 Western blot detection results of different molecular weight Tau proteins in brain tissues of Alzheimer's mice given fibronectin for 28 days. The results showed that there were certain levels of Tau proteins of different molecular weights in the brain homogenate of mice in the blank control group; the levels of Tau proteins of various molecular weights and total Tau proteins in the brain tissues of mice in the drug group were significantly lower than those in the vehicle group, and the statistical analysis of the levels of 35kd, 35-40kd, 40 kd, 54 kd molecular weight Tau proteins and total Tau protein levels in the two groups were 0.174, 0.0406, 0.052, 0.067 and 0.055, respectively. This shows that fibronectin can promote the degradation of Tau proteins in the brain tissues of Alzheimer's model mice.

Figure 13A-B Effects of fibronectin on recombinant human Pro-BDNF in the brain homogenate of Alzheimer's model mice. A is an SDS-PAGE image, and B is the quantitative analysis result of Pro-BDNF band in SDS-PAGE. The results showed that in the brain homogenate of Alzheimer's model mice, the amount of Pro-BDNF in the fibronectin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (* represents P < 0.05, *** represents P < 0.001). This suggests that fibronectin can promote the cleavage of Pro-BDNF in the brain homogenate of Alzheimer's model mice.

Figure 14 A-C Effects of fibronectin on recombinant human Pro-BDNF in the brain homogenate of Alzheimer's model mice. A is a Western blot image, B is the analysis result of the optical density (OD) value of the Pro-BDNF band in Western blot, and C is the analysis result of the optical density (OD) value of the BDNF band in Western blot. The results showed that in the brain homogenate of Alzheimer's model mice, the amount of Pro-BDNF in the fibronectin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (** represents P < 0.01, *** represents P < 0.001); the amount of BDNF in the fibronectin group was significantly higher than that in the vehicle control group, and the difference was extremely significant. These results suggest that fibronectin can promote the cleavage of Pro-BDNF and the formation of mature BDNF in the brain homogenate of Alzheimer's disease model mice.

Figure 15 A-D are the results of immunohistochemical staining of BDNF in the hippocampus of Alzheimer's model mice given fibronectin for 28 days. A is the blank control group, B is the vehicle group, C is the drug group, and D is the average optical density quantitative analysis result. The results showed that the blank control group mice expressed a certain level of BDNF in the hippocampus (indicated by arrows), the expression of BDNF in the hippocampus of the vehicle group mice was significantly lower than that of the blank control group, and the expression of BDNF in the hippocampus of the drug group mice was significantly higher than that of the vehicle group, and the statistical difference was significant (* indicates P < 0.05). This shows that fibronectin can promote the expression of BDNF in the hippocampus of Alzheimer's model mice.

Figure 16 A-C Effects of fibronectin on recombinant human Pro-NGF in brain homogenates of Alzheimer's model mice. A is a Western blot image, B is the analysis result of the optical density (OD) value of the Pro-NGF band in Western blot, and C is the analysis result of the optical density (OD) value of the NGF band in Western blot. The results showed that in the brain homogenates of Alzheimer's model mice, the amount of Pro-NGF in the fibronectin group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** indicates P < 0.001); the amount of NGF in the fibronectin group was significantly higher than that in the vehicle control group, and the difference was significant. This suggests that profibrinolytic enzyme can promote the cleavage of recombinant human Pro-NGF and the formation of mature NGF in the brain homogenate of Alzheimer's disease model mice.

Figure 17 Statistics of the percentage of movement distance in the border zone of Alzheimer's model mice in the field test 28+7 days after administration of fibrinolysinogen. The results showed that the blank control group had a certain percentage of movement distance in the border zone, the percentage of movement distance in the border zone of the mice in the vehicle group was significantly higher than that in the blank control group, and the percentage of movement distance in the border zone of the mice in the drug group was significantly lower than that in the vehicle group, and the statistical difference was close to significant (P=0.08). This suggests that fibrinolysinogen can promote the recovery of anxiety and depression behaviors in Alzheimer's model mice.

Figure 18 Statistical results of the percentage of central area movement distance of Alzheimer's model mice in the field test 28+7 days after administration of fibrinolysinogen. The results showed that the blank control group had a certain percentage of central area movement distance, the percentage of central area movement distance of the vehicle group mice was significantly lower than that of the blank control group, and the percentage of central area movement distance of the drug group mice was significantly higher than that of the vehicle group, and the statistical difference was close to significant (P=0.08). This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 19 Statistics of the total movement distance of Alzheimer's model mice in the elevated plus maze experiment 28+9 days after administration of fibrinolysinogen. The results showed that the blank control group had a certain total movement distance; the movement distance of the vehicle group was significantly greater than that of the blank control group; the total movement distance of the drug group was significantly less than that of the vehicle group, and the statistical difference was extremely significant (* indicates P < 0.05, ** indicates P < 0.01), and was closer to the total movement distance of the blank control group mice. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 20 Statistical results of the closed arm movement distance of Alzheimer's model mice in the elevated plus maze experiment 28+9 days after administration of fibrinolysinogen. The results showed that the mice in the blank control group had a certain closed arm movement distance; the closed arm movement distance of the vehicle group was significantly greater than that of the blank control group; the closed arm movement distance of the drug group was significantly less than that of the vehicle group, and the statistical difference between the two groups was significant (* indicates P < 0.05, ** indicates P < 0.01), and the closed arm movement distance of the drug group was closer to the blank control group. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 21 Statistical results of the percentage of closed arm movement distance in the elevated plus maze experiment of Alzheimer's model mice 28+9 days after administration of fibrinolysinogen. The results showed that the mice in the blank control group had a certain percentage of closed arm movement distance; the percentage of closed arm movement distance in the vehicle group was significantly greater than that in the blank control group; the percentage of closed arm movement distance in the drug group was significantly less than that in the vehicle group, and the statistical difference between the two groups was significant (* indicates P < 0.05), and the percentage of closed arm movement distance in the drug group was closer to the blank control group. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 22 shows the statistical results of the number of closed arm entries in the elevated plus maze experiment of Alzheimer's model mice 28+9 days after administration of fibrinolysinogen. The results showed that the mice in the blank control group had a certain number of closed arm entries; the number of closed arm entries in the vehicle group was significantly more than that in the blank control group; the number of closed arm entries in the drug group was significantly less than that in the vehicle group, and the statistical difference between the two groups was extremely significant (* indicates P < 0.05, ** indicates P < 0.01), and the number of closed arm entries in the drug group was closer to that in the blank control group. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 23 Statistical results of the closed arm time of the elevated plus maze experiment of Alzheimer's model mice 28+9 days after administration of fibrolysinogen. The results showed that the mice in the blank control group had a certain closed arm time, the closed arm stay time of the mice in the vehicle group was significantly shorter than that of the blank control group, and the closed arm time of the mice in the drug group was significantly longer than that of the vehicle group. The statistical difference between the two groups was significant (* indicates P < 0.05, ** indicates P < 0.01), and the closed arm time of the drug group was closer to that of the blank control group.

Figure 24 Statistical results of the percentage of closed arm time in the elevated plus maze experiment of Alzheimer's model mice 28+9 days after administration of fibrolysinogen. The percentage of closed arm time refers to the ratio of the time the mouse stays in the closed arm to the total recorded time. The results showed that the mice in the blank control group had a certain percentage of closed arm time, the percentage of closed arm time in the vehicle group was significantly lower than that in the blank control group, and the percentage of closed arm time in the drug group was significantly higher than that in the vehicle group. The comparison between the two groups was statistically significant (* indicates P < 0.05, ** indicates P < 0.01), and the percentage of closed arm time in the drug group was closer to that in the blank control group.

Figure 25 Statistics of the average movement speed of the closed arms of the elevated plus maze experiment of Alzheimer's model mice 28+9 days after administration of fibrinolysinogen. The results showed that the blank control group had a certain average movement speed of the closed arms, the average movement speed of the closed arms of the vehicle group was significantly greater than that of the blank control group, and the average movement speed of the closed arms of the drug group was significantly less than that of the vehicle group. The statistical difference between the two groups was extremely significant (** indicates P < 0.01), and the average movement speed of the closed arms of the drug group was closer to the blank control group. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 26 Statistics of the percentage of spontaneous alternation reactions in the Y-maze of Alzheimer's model mice 28+9 days after administration of fibrinolysinogen. The results showed that the percentage of spontaneous alternation reactions in the vehicle group was significantly lower than that in the vehicle group, and the percentage of spontaneous alternation reactions in the drug group was significantly higher than that in the vehicle group. The statistical difference between the two groups was significant (* indicates P < 0.05), and was close to that of the blank control mice. This suggests that fibrinolysinogen can promote the recovery of memory function in Alzheimer's model mice.

Figure 27 Statistical results of the closed arm movement distance of Alzheimer's model mice in the elevated plus maze 18 days after administration of fibrinolysinogen. The results showed that the mice in the blank control group had a certain closed arm movement distance; the closed arm movement distance of the vehicle group was significantly less than that of the blank control group; the closed arm movement distance of the drug group was significantly greater than that of the vehicle group, and the statistical difference between the two groups was significant (** indicates P < 0.01, *** indicates P < 0.001), and the closed arm movement distance of the drug group was closer to the blank control group. This suggests that fibrinolysinogen can promote the recovery of anxiety and depressive behavior in Alzheimer's model mice.

Figure 28A-C are representative images of HE staining of the hindbrain tissue of Alzheimer's model mice given fibrolytic enzyme for 8 days. A is the blank control group, B is the vehicle control group, and C is the drug group. The results showed that the morphology of the hippocampal tissue of the blank control group mice was normal; compared with the vehicle group, the morphology of the hippocampal tissue damage in the drug group was significantly improved. This suggests that fibrolytic enzyme can improve the damage of the hippocampus in Alzheimer's model mice.

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Claims (6)

A use of a fibrinolytic enzyme for preparing a drug for promoting the degradation of amyloid protein Aβ40 or Aβ42 in brain tissue, reducing Aβ42 deposition in brain tissue, promoting the degradation of Tau protein in brain tissue, promoting the cleavage of Pro-BDNF in brain tissue to form mature BDNF, promoting the expression of BDNF in brain tissue, promoting the cleavage of Pro-NGF in brain tissue to form mature NGF, and improving brain tissue hippocampal damage, wherein the fibrinolytic enzyme is a protein with at least 90% sequence identity with sequence 2 and a conservative substitution variant that still retains the activity of fibrinolytic enzyme. As for the use of claim 1, the fibrinolysinogen is selected from Glu-fibrinolysinogen and Lys-fibrinolysinogen. The use of any one of claim 1-2, wherein the fibrosinogen is used in combination with one or more other treatment methods or drugs. As for the use of claim 3, the other treatment methods include cell therapy, supportive therapy and physical therapy. As for the use of claim 3, the other drugs are other drugs for treating Alzheimer's disease. The use of any one of claim 1-2, wherein the fibrinolysinogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery) or intramuscular administration.